Anti-tim-3 antibodies and methods of use thereof

ABSTRACT

The instant disclosure provides antibodies that specifically bind to TIM-3 (e.g., human TIM-3) and antagonize TIM-3 function. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/806,299, filed Mar. 2, 2020, which is a continuation of U.S. patentapplication Ser. No. 15/606,148, filed May 26, 2017, now U.S. Pat. No.10,639,368, which claims the benefit of U.S. Provisional ApplicationNos: 62/342,610, filed May 27, 2016; and 62/420,276, filed Nov. 10,2016, each of which is incorporated by reference herein in its entirety.

1. FIELD

The instant disclosure relates to antibodies that specifically bind toTIM-3 (e.g., human TIM-3) and methods of using the same.

2. BACKGROUND

The protein T cell immunoglobulin and mucin domain-3 (TIM-3) is a type Imembrane protein in the immunoglobulin (Ig) superfamily. It has anextracellular Ig variable-like (IgV) domain, an extracellular mucin-likedomain, and a cytoplasmic domain with six conserved tyrosine residues(Monney et al. (2002) Nature 415:536-41). TIM-3 is expressed onactivated T-helper type 1 (Th1) and CD8⁺ T (Tc1) lymphocytes, somemacrophages (Monney et al. (2002) Nature 415:536-41), activated naturalkiller (NK) cells (Ndhlovu et al. (2012) Blood 119(16):3734-43), andIL-17-producing Th17 cells (Nakae et al. (2007) J Leukoc Biol 81:1258-68).

Studies have shown that TIM-3 functions to inhibit T cell, myeloid cell,and NK cell-mediated responses and to promote immunological tolerance.For example, TIM-3 IgV peptide fused with an immunoglobulin domain,which binds to and neutralizes TIM-3 ligands, caused hyperproliferationof Th1 cells and Th1 cytokine release in immunized mice (Sabatos et al.(2003) Nat Immunol 4:1102-10). Indeed, in vivo administration of ananti-TIM-3 antibody enhanced the pathological severity of experimentalautoimmune encephalomyelitis, an animal model of multiple sclerosis(Monney et al. (2002) Nature 415:536-41). Moreover, TIM-3 expression isupregulated in CD8⁺ T cells in cancer patients. For example,approximately 30% of NY-ESO-1-specific CD8+ T cells in patients withadvanced melanoma exhibit upregulation of TIM-3 expression (Fourcade etal. (2010) J Exp Med 207:2175-86).

Given the apparent role of human TIM-3 in modulating immune responses,therapeutic agents designed to antagonize TIM-3 signaling hold greatpromise for the treatment of diseases that involve TIM-3-mediated immunesuppression.

3. SUMMARY

The instant disclosure provides antibodies that specifically bind toTIM-3 (e.g., human TIM-3) and antagonize TIM-3 function, e.g.,TIM-3-mediated immune suppression. Also provided are pharmaceuticalcompositions comprising these antibodies, nucleic acids encoding theseantibodies, expression vectors and host cells for making theseantibodies, and methods of treating a subject using these antibodies.The antibodies disclosed herein are particularly useful for increasing Tcell activation in response to an antigen (e.g., a tumor antigen or aninfectious disease antigen) and/or decreasing Treg-mediated immunesuppression, and hence for treating cancer in a subject or treating orpreventing an infectious disease in a subject.

Accordingly, in one aspect, the instant disclosure provides an antibodyor isolated antibody comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2 and CDRH3 and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2 and CDRL3, wherein:

-   -   (a) CDRH1 comprises the amino acid sequence of X₁X₂X₃X₄X₅S (SEQ        ID NO: 48), wherein        -   X₁ is R, S, A, G, K, M, or T,        -   X₂ is Q, S, A, G, R, or T,        -   X₃ is N, Y, G, or Q,        -   X₄ is A or Q, and        -   X₅ is W, M, A, S, or T;    -   (b) CDRH2 comprises the amino acid sequence of WVSAISGSGGSTY        (SEQ ID NO: 2);    -   (c) CDRH3 comprises the amino acid sequence of AKGGDYGGNYFD (SEQ        ID NO: 3);    -   (d) CDRL1 comprises the amino acid sequence of X₁ASQSVX₂SSYLA        (SEQ ID NO: 52), wherein        -   X₁ is R or G, and        -   X₂ is absent or S;    -   (e) CDRL2 comprises the amino acid sequence of X₁ASX₂RAT (SEQ ID        NO: 53), wherein        -   X₁ is D or G, and        -   X₂ is N, S, or T; and    -   (f) CDRL3 comprises the amino acid sequence of QQYGSSPX₁T (SEQ        ID NO: 54), wherein X₁ is L or I.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, the antibodycomprising a heavy chain variable region comprising complementaritydetermining regions CDRH1, CDRH2 and CDRH3 and a light chain variableregion comprising complementarity determining regions CDRL1, CDRL2 andCDRL3, wherein:

-   -   (a) CDRH1 comprises the amino acid sequence of X₁X₂X₃X₄X₅S (SEQ        ID NO: 48), wherein        -   X₁ is R, S, A, G, K, M, or T,        -   X₂ is Q, S, A, G, R, or T,        -   X₃ is N, Y, G, or Q,        -   X₄ is A or Q, and        -   X₅ is W, M, A, S, or T;    -   (b) CDRH2 comprises the amino acid sequence of WVSAISGSGGSTY        (SEQ ID NO: 2);    -   (c) CDRH3 comprises the amino acid sequence of AKGGDYGGNYFD (SEQ        ID NO: 3);    -   (d) CDRL1 comprises the amino acid sequence of X₁ASQSVX₂SSYLA        (SEQ ID NO: 52), wherein        -   X₁ is R or G, and        -   X₂ is absent or S;    -   (e) CDRL2 comprises the amino acid sequence of X₁ASX₂RAT (SEQ ID        NO: 53), wherein        -   X₁ is D or G, and        -   X₂ is N, S, or T; and    -   (f) CDRL3 comprises the amino acid sequence of QQYGSSPX₁T (SEQ        ID NO: 54), wherein X₁ is L or I.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region havingcomplementarity determining regions CDRH1, CDRH2 and CDRH3 and a lightchain variable region having complementarity determining regions CDRL1,CDRL2 and CDRL3, wherein the antibody is internalized upon binding tocells expressing human TIM-3, and wherein CDRH3 comprises the amino acidsequence of AKGGDYGGNYFD (SEQ ID NO: 3).

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, the antibodycomprising a heavy chain variable region having complementaritydetermining regions CDRH1, CDRH2 and CDRH3 and a light chain variableregion having complementarity determining regions CDRL1, CDRL2 andCDRL3, wherein the antibody is internalized upon binding to cellsexpressing human TIM-3, and wherein CDRH3 comprises the amino acidsequence of AKGGDYGGNYFD (SEQ ID NO: 3).

In certain embodiments:

-   -   (a) CDRH1 comprises the amino acid sequence of X₁X₂X₃X₄X₅S (SEQ        ID NO: 48), wherein        -   X₁ is R, S, A, G, K, M, or T,        -   X₂ is Q, S, A, G, R, or T,        -   X₃ is N, Y, G, or Q,        -   X₄ is A or Q, and        -   X₅ is W, M, A, S, or T;    -   (b) CDRH2 comprises the amino acid sequence of WVSAISGSGGSTY        (SEQ ID NO: 2);    -   (c) CDRL1 comprises the amino acid sequence of X₁ASQSVX₂SSYLA        (SEQ ID NO: 52), wherein        -   X₁ is R or G, and        -   X₂ is absent or S;    -   (d) CDRL2 comprises the amino acid sequence of X₁ASX₂RAT (SEQ ID        NO: 53), wherein        -   X₁ is D or G, and        -   X₂ is N, S, or T; and    -   (e) CDRL3 comprises the amino acid sequence of QQYGSSPX₁T (SEQ        ID NO: 54), wherein X₁ is L or I.

In certain embodiments, CDRH1 comprises the amino acid sequence ofX₁X₂NAWS (SEQ ID NO: 49), wherein: X₁ is R or A; and X₂ is Q or R. Incertain embodiments, CDRH1 comprises the amino acid sequence ofX₁X₂GQX₃S (SEQ ID NO: 50), wherein: X₁ is K, M, or G; X₂ is A or S; andX₃ is S or T. In certain embodiments, CDRH1 comprises the amino acidsequence of X₁X₂QQAS (SEQ ID NO: 51), wherein: X₁ is S, R, T, or G; andX₂ is A, S, T, or G. In certain embodiments, CDRH1 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1 and4-12.

In certain embodiments, CDRL1 comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 13-16. In certain embodiments,CDRL2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 17-21. In certain embodiments, CDRL3 comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:22 and 23.

In certain embodiments, CDRH1, CDRH2 and CDRH3 comprise the CDRH1, CDRH2and CDRH3 amino acid sequences, respectively, set forth in SEQ ID NOs:1, 2, and 3; 4, 2, and 3; 5, 2, and 3; 6, 2, and 3; 7, 2, and 3; 8, 2,and 3; 9, 2, and 3; 10, 2, and 3; 11, 2, and 3; or 12, 2, and 3.

In certain embodiments, CDRL1, CDRL2 and CDRL3 comprise the CDRL1, CDRL2and CDRL3 amino acid sequences, respectively, set forth in SEQ ID NOs:13, 17, and 22; 14, 17, and 22; 15, 18, and 22; 14, 19, and 22; 14, 20,and 22; 14, 21, and 22; 16, 20, and 22; or 14, 17, and 23.

In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2, 3, 14,21, and 22; 4, 2, 3, 14, 21, and 22; 5, 2, 3, 14, 21, and 22; 6, 2, 3,14, 21, and 22; 7, 2, 3, 14, 21, and 22; 8, 2, 3, 14, 21, and 22; 9, 2,3, 14, 21, and 22; 10, 2, 3, 14, 21, and 22; 11, 2, 3, 14, 21, and 22;or 12, 2, 3, 14, 21, and 22, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2 and CDRH3, and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, andCDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2,3, 14, 21, and 22, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, comprising aheavy chain variable region comprising complementarity determiningregions CDRH1, CDRH2 and CDRH3, and a light chain variable regioncomprising complementarity determining regions CDRL1, CDRL2 and CDRL3,wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the aminoacid sequences set forth in SEQ ID NOs: 1, 2, 3, 14, 21, and 22,respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2 and CDRH3, and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, andCDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 5, 2,3, 14, 21, and 22, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, comprising aheavy chain variable region comprising complementarity determiningregions CDRH1, CDRH2 and CDRH3, and a light chain variable regioncomprising complementarity determining regions CDRL1, CDRL2 and CDRL3,wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the aminoacid sequences set forth in SEQ ID NOs: 5, 2, 3, 14, 21, and 22,respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2 and CDRH3, and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, andCDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 9, 2,3, 14, 21, and 22, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, comprising aheavy chain variable region comprising complementarity determiningregions CDRH1, CDRH2 and CDRH3, and a light chain variable regioncomprising complementarity determining regions CDRL1, CDRL2 and CDRL3,wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the aminoacid sequences set forth in SEQ ID NOs: 9, 2, 3, 14, 21, and 22,respectively.

In another aspect, the instant disclosure provides an antibody orisolated comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2 and CDRH3, and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, andCDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 1, 2,3, 15, 18, and 22, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, comprising aheavy chain variable region comprising complementarity determiningregions CDRH1, CDRH2 and CDRH3, and a light chain variable regioncomprising complementarity determining regions CDRL1, CDRL2 and CDRL3,wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the aminoacid sequences set forth in SEQ ID NOs: 1, 2, 3, 15, 18, and 22,respectively.

In certain embodiments, the antibody is internalized upon binding tocells expressing human TIM-3.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, wherein theantibody is internalized upon binding to cells expressing human TIM-3.

In certain embodiments, a lower percentage of the cells expressing humanTIM-3 survive in the presence of the antibody than in the presence ofpab1944w (IgG₁ N297A) in an assay comprising the following steps: (a)plating the cells expressing human TIM-3 at 2×10⁴ cells per well in atissue culture plate; (b) adding 1111 ng/ml of αHFc-NC-DM1 and 1111ng/ml of the antibody or pab1944w (IgG₁ N297A) at a final volume of 100μl/well; (c) incubating at 37° C. and 5% CO₂ for 72 hours; (d) measuringsurvival of the cells expressing human TIM-3; and (e) calculatingpercentage of cell survival relative to untreated cells expressing humanTIM-3. In certain embodiments, the percentage of cell survival in thepresence of the antibody is at least 50% lower than the percentage ofcell survival in the presence of pab1944w (IgG₁ N297A). In certainembodiments, the cells expressing human TIM-3 are Kasumi-3 cells. Incertain embodiments, the cells expressing human TIM-3 are Kasumi-3 cells(ATCC® CRL-2725™). In certain embodiments, the cells expressing humanTIM-3 are Jurkat cells engineered to express human TIM-3.

In certain embodiments, a lower percentage of the cells expressing humanTIM-3 survive in the presence of the antibody than in the presence ofHum11 (IgG₄ S228P) in an assay comprising the following steps: (a)plating the cells expressing human TIM-3 at 2×10⁴ cells per well in atissue culture plate; (b) adding 1111 ng/ml of αHFc-NC-DM1 and 1111ng/ml of the antibody or Hum11 (IgG₄ S228P) at a final volume of 100μl/well; (c) incubating at 37° C. and 5% CO₂ for 72 hours; (d) measuringsurvival of the cells expressing human TIM-3; and (e) calculatingpercentage of cell survival relative to untreated cells expressing humanTIM-3. In certain embodiments, the percentage of cell survival in thepresence of the antibody is at least 50% lower than the percentage ofcell survival in the presence of Hum11 (IgG₄ S228P). In certainembodiments, the cells expressing human TIM-3 are Kasumi-3 cells. Incertain embodiments, the cells expressing human TIM-3 are Kasumi-3 cells(ATCC® CRL-2725™). In certain embodiments, the cells expressing humanTIM-3 are Jurkat cells engineered to express human TIM-3.

In certain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 55. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an amino acid sequence which is at least 75%, 80%, 85%, 90%,95%, or 100% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 24-35. In certain embodiments, the heavy chainvariable region comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 24-35. In certain embodiments, the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 25. Incertain embodiments, the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO: 28. In certain embodiments, the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 32. Incertain embodiments, the N-terminal glutamate (E) residue of a heavychain variable region of an antibody as described herein is replacedwith a pyroglutamate (pE) residue.

In certain embodiments, the antibody comprises a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 56. In certainembodiments, the antibody comprises a light chain variable regioncomprising an amino acid sequence which is at least 75%, 80%, 85%, 90%,95%, or 100% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 36-47. In certain embodiments, the light chainvariable region comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 36-47. In certain embodiments, the light chainvariable region comprises the amino acid sequence of SEQ ID NO: 46. Incertain embodiments, the N-terminal glutamate (E) residue of a lightchain variable region of an antibody as described herein is replacedwith a pyroglutamate (pE) residue.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:24-35. In certain embodiments, the heavy chain variable region comprisesthe amino acid sequence of SEQ ID NO: 25. In certain embodiments, theheavy chain variable region comprises the amino acid sequence of SEQ IDNO: 28. In certain embodiments, the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO: 32. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 58. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:61. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 65. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 70, 71, 72, 73, 74 or 75. In certainembodiments, the N-terminal glutamate (E) residue of a heavy chain of anantibody as described herein is replaced with a pyroglutamate (pE)residue.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, the antibodycomprising a heavy chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 24-35. Incertain embodiments, the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO: 25. In certain embodiments, the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 28. Incertain embodiments, the heavy chain variable region comprises the aminoacid sequence of SEQ ID NO: 32. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:58. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 61. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 65. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:70, 71, 72, 73, 74 or 75.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a light chain variable region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:36-47. In certain embodiments, the light chain variable region comprisesthe amino acid sequence of SEQ ID NO: 46. In certain embodiments, theantibody comprises a light chain comprising the amino acid sequence ofSEQ ID NO: 69. In certain embodiments, the antibody comprises a lightchain comprising the amino acid sequence of SEQ ID NO: 76 or 77. Incertain embodiments, the N-terminal glutamate (E) residue of a lightchain of an antibody as described herein is replaced with apyroglutamate (pE) residue.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, the antibodycomprising a light chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 36-47. Incertain embodiments, the light chain variable region comprises the aminoacid sequence of SEQ ID NO: 46. In certain embodiments, the antibodycomprises a light chain comprising the amino acid sequence of SEQ ID NO:69. In certain embodiments, the antibody comprises a light chaincomprising the amino acid sequence of SEQ ID NO: 76 or 77.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region and thelight chain variable region, respectively, comprise the amino acidsequences set forth in SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34and 46; or 35 and 46. In certain embodiments, the heavy chain variableregion and the light chain variable region, respectively, comprise theamino acid sequences set forth in SEQ ID NOs: 25 and 46. In certainembodiments, the heavy chain variable region and the light chainvariable region, respectively, comprise the amino acid sequences setforth in SEQ ID NOs: 28 and 46. In certain embodiments, the heavy chainvariable region and the light chain variable region, respectively,comprise the amino acid sequences set forth in SEQ ID NOs: 32 and 46. Incertain embodiments, the N-terminal glutamate (E) residue of a heavychain variable region of an antibody as described herein is replacedwith a pyroglutamate (pE) residue and/or the N-terminal glutamate (E)residue of a light chain variable region of the antibody is replacedwith a pyroglutamate (pE) residue.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain variable region and a lightchain variable region, wherein the amino acid sequences of the heavychain variable region and the light chain variable region, respectively,consist of the amino acid sequences set forth in SEQ ID NOs: 24 and 36;24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32and 46; 33 and 46; 34 and 46; or 35 and 46. In certain embodiments, theamino acid sequences of the heavy chain variable region and the lightchain variable region, respectively, consist of the amino acid sequencesset forth in SEQ ID NOs: 25 and 46. In certain embodiments, the aminoacid sequences of the heavy chain variable region and the light chainvariable region, respectively, consist of the amino acid sequences setforth in SEQ ID NOs: 28 and 46. In certain embodiments, the amino acidsequences of the heavy chain variable region and the light chainvariable region, respectively, consist of the amino acid sequences setforth in SEQ ID NOs: 32 and 46.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, the antibodycomprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region and the light chainvariable region, respectively, comprise the amino acid sequences setforth in SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and46. In certain embodiments, the heavy chain variable region and thelight chain variable region, respectively, comprise the amino acidsequences set forth in SEQ ID NOs: 25 and 46. In certain embodiments,the heavy chain variable region and the light chain variable region,respectively, comprise the amino acid sequences set forth in SEQ ID NOs:28 and 46. In certain embodiments, the heavy chain variable region andthe light chain variable region, respectively, comprise the amino acidsequences set forth in SEQ ID NOs: 32 and 46.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, the antibodycomprising a heavy chain variable region and a light chain variableregion, wherein the amino acid sequences of the heavy chain variableregion and the light chain variable region, respectively, consist of theamino acid sequences set forth in SEQ ID NOs: 24 and 36; 24 and 38; 26and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33and 46; 34 and 46; or 35 and 46. In certain embodiments, the amino acidsequences of the heavy chain variable region and the light chainvariable region, respectively, consist of the amino acid sequences setforth in SEQ ID NOs: 25 and 46. In certain embodiments, the amino acidsequences of the heavy chain variable region and the light chainvariable region, respectively, consist of the amino acid sequences setforth in SEQ ID NOs: 28 and 46. In certain embodiments, the amino acidsequences of the heavy chain variable region and the light chainvariable region, respectively, consist of the amino acid sequences setforth in SEQ ID NOs: 32 and 46.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 58, and a light chain comprising the amino acidsequence of SEQ ID NO: 69.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 58, and a light chain comprising the amino acid sequence of SEQ IDNO: 69.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 61, and a light chain comprising the amino acidsequence of SEQ ID NO: 69.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 61, and a light chain comprising the amino acid sequence of SEQ IDNO: 69.

In another aspect, the instant disclosure provides an antibody orisolated antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 65, and a light chain comprising the amino acidsequence of SEQ ID NO: 69.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 65, and a light chain comprising the amino acid sequence of SEQ IDNO: 69.

In certain embodiments, the N-terminal glutamate (E) residue of a heavychain of an antibody as described herein is replaced with apyroglutamate (pE) residue and/or the N-terminal glutamate (E) residueof a light chain of the antibody is replaced with a pyroglutamate (pE)residue.

In certain embodiments, the antibody comprises a heavy chain variableregion having an amino acid sequence derived from a human IGHV3-23germline sequence. In certain embodiments, the antibody comprises alight chain variable region having an amino acid sequence derived from ahuman germline sequence selected from the group consisting of IGKV1-27,IGKV3-11, IGKV3-20, and IGKV3D-20.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, the antibodycomprising a heavy chain variable region having an amino acid sequencederived from a human IGHV3-23 germline sequence, and a light chainvariable region having an amino acid sequence derived from a humangermline sequence selected from the group consisting of IGKV1-27,IGKV3-11, IGKV3-20, and IGKV3D-20.

In certain embodiments, the antibody comprises a heavy chain constantregion selected from the group consisting of human IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂. In certain embodiments, the heavy chain constantregion is IgG₁. In certain embodiments, the amino acid sequence of IgG₁comprises a N297A mutation, numbered according to the EU numberingsystem. In certain embodiments, the antibody comprises a heavy chainconstant region comprising the amino acid sequence of SEQ ID NO: 72. Incertain embodiments, the amino acid sequence of IgG₁ comprises a N297Qmutation, numbered according to the EU numbering system. In certainembodiments, the IgG₁ is non-fucosylated IgG₁. In certain embodiments,the heavy chain constant region is IgG₄. In certain embodiments, theamino acid sequence of IgG₄ comprises a S228P mutation, numberedaccording to the EU numbering system. In certain embodiments, theantibody comprises a heavy chain constant region comprising the aminoacid sequence of SEQ ID NO: 74.

In certain embodiments, the antibody comprises a light chain constantregion selected from the group consisting of human IgGκ and IgGλ. Incertain embodiments, the light chain constant region is IgGκ. In certainembodiments, the antibody comprises a light chain constant regioncomprising the amino acid sequence of SEQ ID NO: 76. In certainembodiments, the light chain constant region is IgGλ.

In another aspect, the instant disclosure provides an antibody orisolated antibody that cross-competes for binding to human TIM-3 with anantibody as disclosed herein. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that cross-competesfor binding to human TIM-3 with an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 55 and 56, respectively. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that cross-competesfor binding to human TIM-3 with an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 25 and 46, respectively. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that cross-competesfor binding to human TIM-3 with an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 28 and 46, respectively. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that cross-competesfor binding to human TIM-3 with an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 32 and 46, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody that binds to the same epitope of human TIM-3 as anantibody disclosed herein. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that binds to thesame epitope of human TIM-3 as an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 55 and 56, respectively. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that binds to thesame epitope of human TIM-3 as an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 25 and 46, respectively. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that binds to thesame epitope of human TIM-3 as an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 28 and 46, respectively. In certain embodiments, the instantdisclosure provides an antibody or isolated antibody that binds to thesame epitope of human TIM-3 as an antibody comprising the heavy andlight chain variable region amino acid sequences set forth in SEQ IDNOs: 32 and 46, respectively.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, wherein theantibody specifically binds to a variant TIM-3 protein having the aminoacid sequence of SEQ ID NO: 101 with a lower affinity than to awild-type TIM-3 protein having the amino acid sequence of SEQ ID NO: 79.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanTIM-3 as any antibody of the present invention. In one embodiment, theantibody specifically binds to a variant TIM-3 protein having the aminoacid sequence of SEQ ID NO: 101 with a lower affinity than to awild-type TIM-3 protein having the amino acid sequence of SEQ ID NO: 79.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, wherein theantibody does not specifically bind to a variant TIM-3 protein havingthe amino acid sequence of SEQ ID NO: 101.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanTIM-3 as any antibody of the present invention. In one embodiment, theantibody does not specifically bind to a variant TIM-3 protein havingthe amino acid sequence of SEQ ID NO: 101.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, wherein thebinding between the antibody and a variant TIM-3 protein having theamino acid sequence of SEQ ID NO: 101 is substantially weakened relativeto the binding between the antibody and a wild-type TIM-3 protein havingthe amino acid sequence of SEQ ID NO: 79.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanTIM-3 as any antibody of the present invention. In one embodiment, thebinding between the antibody and a variant TIM-3 protein having theamino acid sequence of SEQ ID NO: 101 is substantially weakened relativeto the binding between the antibody and a wild-type TIM-3 protein havingthe amino acid sequence of SEQ ID NO: 79.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to human TIM-3, wherein theantibody exhibits, as compared to binding to a wild-type TIM-3 proteinhaving the amino acid sequence of SEQ ID NO: 79, reduced or absentbinding to a variant TIM-3 protein having the amino acid sequence of SEQID NO: 101.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanTIM-3 as any antibody of the present invention. In one embodiment, theantibody exhibits, as compared to binding to a wild-type TIM-3 proteinhaving the amino acid sequence of SEQ ID NO: 79, reduced or absentbinding to a variant TIM-3 protein having the amino acid sequence of SEQID NO: 101.

In another aspect, the instant disclosure provides an antibody orisolated antibody that binds, e.g., specifically binds, to an epitope ofhuman TIM-3. In certain embodiments, the antibody binds to residue 40 ofSEQ ID NO: 79.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanTIM-3 as any antibody of the present invention. In certain embodiments,the antibody binds to residue 40 of SEQ ID NO: 79.

In another aspect, the instant disclosure provides an antibody orisolated antibody that binds, e.g., specifically binds, to an epitope ofhuman TIM-3. In certain embodiments, the antibody binds to an epitopelocated within a region of human TIM-3 consisting of the amino acidsequence of SEQ ID NO: 93. In certain embodiments, the antibody binds toan epitope located within a region of human TIM-3 consisting of theamino acid sequence of SEQ ID NO: 94. In certain embodiments, theantibody binds to an epitope located within a region of human TIM-3consisting of the amino acid sequence of SEQ ID NO: 95. In certainembodiments, the antibody binds to an epitope located within a region ofhuman TIM-3 consisting of the amino acid sequence of SEQ ID NO: 96. Incertain embodiments, the antibody binds to an epitope located within aregion of human TIM-3 consisting of the amino acid sequence of SEQ IDNO: 97. In certain embodiments, the antibody binds to an epitope locatedwithin a region of human TIM-3 consisting of the amino acid sequence ofSEQ ID NO: 98. In certain embodiments, the antibody binds to an epitopelocated within a region of human TIM-3 consisting of the amino acidsequence of SEQ ID NO: 99. In certain embodiments, the antibody binds toan epitope located within a region of human TIM-3 consisting of theamino acid sequence of SEQ ID NO: 100.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanTIM-3 as any antibody of the present invention. In certain embodiments,the antibody binds to an epitope located within a region of human TIM-3consisting of the amino acid sequence of SEQ ID NO: 93. In certainembodiments, the antibody binds to an epitope located within a region ofhuman TIM-3 consisting of the amino acid sequence of SEQ ID NO: 94. Incertain embodiments, the antibody binds to an epitope located within aregion of human TIM-3 consisting of the amino acid sequence of SEQ IDNO: 95. In certain embodiments, the antibody binds to an epitope locatedwithin a region of human TIM-3 consisting of the amino acid sequence ofSEQ ID NO: 96. In certain embodiments, the antibody binds to an epitopelocated within a region of human TIM-3 consisting of the amino acidsequence of SEQ ID NO: 97. In certain embodiments, the antibody binds toan epitope located within a region of human TIM-3 consisting of theamino acid sequence of SEQ ID NO: 98. In certain embodiments, theantibody binds to an epitope located within a region of human TIM-3consisting of the amino acid sequence of SEQ ID NO: 99. In certainembodiments, the antibody binds to an epitope located within a region ofhuman TIM-3 consisting of the amino acid sequence of SEQ ID NO: 100.

In another aspect, the instant disclosure provides an antibody that,when bound to a human TIM-3 protein or fragment thereof comprising theamino acid sequence of SEQ ID NO: 102, reduces hydrogen/deuteriumexchange in a region consisting of the amino acid sequence set forth inSEQ ID NO: 93 relative to hydrogen/deuterium exchange in the regionconsisting of the amino acid sequence set forth in SEQ ID NO: 93 in theabsence of the antibody, as determined by a hydrogen/deuterium assay. Inanother aspect, the instant disclosure provides an antibody that, whenbound to a human TIM-3 protein or fragment thereof comprising the aminoacid sequence of SEQ ID NO: 102, reduces hydrogen/deuterium exchange ina region consisting of the amino acid sequence set forth in SEQ ID NO:94 relative to hydrogen/deuterium exchange in the region consisting ofthe amino acid sequence set forth in SEQ ID NO: 94 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In anotheraspect, the instant disclosure provides an antibody that, when bound toa human TIM-3 protein or fragment thereof comprising the amino acidsequence of SEQ ID NO: 102, reduces hydrogen/deuterium exchange in aregion consisting of the amino acid sequence set forth in SEQ ID NO: 95relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 95 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In anotheraspect, the instant disclosure provides an antibody that, when bound toa human TIM-3 protein or fragment thereof comprising the amino acidsequence of SEQ ID NO: 102, reduces hydrogen/deuterium exchange in aregion consisting of the amino acid sequence set forth in SEQ ID NO: 96relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 96 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In anotheraspect, the instant disclosure provides an antibody that, when bound toa human TIM-3 protein or fragment thereof comprising the amino acidsequence of SEQ ID NO: 102, reduces hydrogen/deuterium exchange in aregion consisting of the amino acid sequence set forth in SEQ ID NO: 97relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 97 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In anotheraspect, the instant disclosure provides an antibody that, when bound toa human TIM-3 protein or fragment thereof comprising the amino acidsequence of SEQ ID NO: 102, reduces hydrogen/deuterium exchange in aregion consisting of the amino acid sequence set forth in SEQ ID NO: 98relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 98 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In someembodiments, the reduction in hydrogen/deuterium exchange is measuredusing hydrogen-deuterium exchange (HDX), for example as described hereinin the examples.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanTIM-3 as any antibody of the present invention. In certain embodiments,the antibody, when bound to a human TIM-3 protein or fragment thereofcomprising the amino acid sequence of SEQ ID NO: 102, reduceshydrogen/deuterium exchange in a region consisting of the amino acidsequence set forth in SEQ ID NO: 93 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 93 in the absence of the antibody, as determined by ahydrogen/deuterium assay. In certain embodiments, the antibody, whenbound to a human TIM-3 protein or fragment thereof comprising the aminoacid sequence of SEQ ID NO: 102, reduces hydrogen/deuterium exchange ina region consisting of the amino acid sequence set forth in SEQ ID NO:94 relative to hydrogen/deuterium exchange in the region consisting ofthe amino acid sequence set forth in SEQ ID NO: 94 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In certainembodiments, the antibody, when bound to a human TIM-3 protein orfragment thereof comprising the amino acid sequence of SEQ ID NO: 102,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 95 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 95 in the absence of the antibody, as determined by ahydrogen/deuterium assay. In certain embodiments, the antibody, whenbound to a human TIM-3 protein or fragment thereof comprising the aminoacid sequence of SEQ ID NO: 102, reduces hydrogen/deuterium exchange ina region consisting of the amino acid sequence set forth in SEQ ID NO:96 relative to hydrogen/deuterium exchange in the region consisting ofthe amino acid sequence set forth in SEQ ID NO: 96 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In certainembodiments, the antibody, when bound to a human TIM-3 protein orfragment thereof comprising the amino acid sequence of SEQ ID NO: 102,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 97 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 97 in the absence of the antibody, as determined by ahydrogen/deuterium assay. In certain embodiments, the antibody, whenbound to a human TIM-3 protein or fragment thereof comprising the aminoacid sequence of SEQ ID NO: 102, reduces hydrogen/deuterium exchange ina region consisting of the amino acid sequence set forth in SEQ ID NO:98 relative to hydrogen/deuterium exchange in the region consisting ofthe amino acid sequence set forth in SEQ ID NO: 98 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In someembodiments, the reduction in hydrogen/deuterium exchange is measuredusing hydrogen-deuterium exchange (HDX), for example as described hereinin the examples.

In another aspect, the instant disclosure provides an antibody orisolated antibody that binds, e.g., specifically binds, to the sameepitope of human TIM-3 as any antibody of the present invention, whereinthe epitope is determined by hydrogen-deuterium exchange (HDX), forexample as described in the examples, by Pepscan analysis, for exampleas described in the examples, or by Alanine scanning, for example asdescribed in the examples.

In certain embodiments, the antibody comprises a human IgG heavy chainconstant region that is a variant of a wild type human IgG heavy chainconstant region, wherein the variant human IgG heavy chain constantregion binds to a human Fc gamma receptor with lower affinity than thewild type human IgG heavy chain constant region binds to the human Fcgamma receptor. In certain embodiments, the human Fc gamma receptor isselected from the group consisting of FcγRI, FcγRII, and FcγRIII. Incertain embodiments, the variant human IgG heavy chain constant regionis an IgG₁ constant region comprising a N297A mutation.

In certain embodiments, the antibody is a human antibody. In certainembodiments, the antibody is antagonistic to human TIM-3. In certainembodiments, the antibody deactivates, reduces, or inhibits an activityof human TIM-3. In certain embodiments, the antibody inhibits binding ofhuman TIM-3 to phosphatidylserine. In certain embodiments, the antibodyinduces IFNγ production by peripheral blood mononuclear cells (PBMCs)stimulated with staphylococcal enterotoxin A (SEA). In certainembodiments, the antibody induces IFNγ or TNFα production by tumorinfiltrating lymphocytes (TILs) stimulated with anti-CD3 and anti-CD28antibodies.

In certain embodiments, the antibody is internalized upon binding tocells expressing human TIM-3.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein conjugated to a cytotoxic agent.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein conjugated to a cytostatic agent.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein conjugated to a toxin.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein conjugated to a radionuclide.

In another aspect, the instant disclosure provides an antibody orisolated antibody as disclosed herein conjugated to a detectable label.

In another aspect, the instant disclosure provides a pharmaceuticalcomposition comprising an antibody as disclosed herein and apharmaceutically acceptable carrier or excipient.

In another aspect, the instant disclosure provides a polynucleotide orisolated polynucleotide encoding a heavy and/or light chain of anantibody as disclosed herein. In another aspect, the instant disclosureprovides a vector comprising the polynucleotide. In another aspect, theinstant disclosure provides a recombinant host cell comprising thepolynucleotide. In another aspect, the instant disclosure provides arecombinant host cell comprising the vector. In another aspect, theinstant disclosure provides a method of producing an antibody asdisclosed herein, the method comprising culturing the host cell so thatthe polynucleotide is expressed and the antibody is produced. In oneembodiment, the method is an in vitro method.

In one embodiment, the present invention relates to an antibody of theinvention, or a pharmaceutical composition of the invention, or apolynucleotide of the invention, or a vector of the invention, or arecombinant host cell of the invention for use as a medicament.

In one embodiment, the present invention relates to an antibody of theinvention, or a pharmaceutical composition of the invention, or apolynucleotide of the invention, or a vector of the invention, or arecombinant host cell of the invention for use as a diagnostic.

In another aspect, the instant disclosure provides a method ofincreasing T cell activation in response to an antigen in a subject, themethod comprising administering to the subject an effective amount of anantibody or pharmaceutical composition as disclosed herein. In anotheraspect, the instant disclosure provides a method of treating cancer in asubject, the method comprising administering to the subject an effectiveamount of an antibody or pharmaceutical composition as disclosed herein.In certain embodiments of the foregoing methods, the antibody orpharmaceutical composition is administered subcutaneously. In certainembodiments of the foregoing methods, the antibody or pharmaceuticalcomposition is administered intravenously. In certain embodiments of theforegoing methods, the antibody or pharmaceutical composition isadministered intratumorally. In certain embodiments of the foregoingmethods, the antibody or pharmaceutical composition is delivered to atumor draining lymph node. In certain embodiments of the foregoingmethods, the antibody or pharmaceutical composition is administeredintra-arterially.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for increasingT cell activation in response to an antigen.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for increasingT cell activation in response to an antigen in a subject.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for increasingT cell activation in response to an antigen in a subject comprisingadministering to the subject an effective amount of an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the invention.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for thetreatment of cancer.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for thetreatment of cancer in a subject.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention for use in a method for thetreatment of cancer in a subject comprising administering to the subjectan effective amount of an antibody, polynucleotide, vector, recombinanthost cell, and/or pharmaceutical composition of the invention.

In one embodiment of an antibody, polynucleotide, vector, recombinanthost cell, and/or pharmaceutical composition for use of the presentinvention, the antibody, polynucleotide, vector, recombinant host cell,and/or pharmaceutical composition is administered subcutaneously orintravenously. In another embodiment of an antibody, polynucleotide,vector, recombinant host cell, and/or pharmaceutical composition for useof the present invention, the antibody, polynucleotide, vector,recombinant host cell, and/or pharmaceutical composition is administeredintratumorally or intra-arterially.

In certain embodiments, the foregoing methods further compriseadministering an additional therapeutic agent to the subject. Therefore,in one embodiment of an antibody, polynucleotide, vector, recombinanthost cell, and/or pharmaceutical composition for use in a method of thepresent invention, the method further comprises administering anadditional therapeutic agent to the subject.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent for use as a medicament.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent for use in a method for the treatment of cancer.

In one aspect, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent.

In certain embodiments, the additional therapeutic agent is achemotherapeutic. In certain embodiments, the additional therapeuticagent is a radiotherapeutic.

In certain embodiments, the additional therapeutic agent is a checkpointtargeting agent. In certain embodiments, the checkpoint targeting agentis selected from the group consisting of an antagonist anti-PD-1antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2antibody, an antagonist anti-CTLA-4 antibody, an antagonist anti-TIM-3antibody, an antagonist anti-LAG-3 antibody, an antagonist anti-CEACAM1antibody, an agonist anti-CD137 antibody, an antagonist anti-TIGITantibody, an antagonist anti-VISTA antibody, an agonist anti-GITRantibody, and an agonist anti-OX40 antibody. In certain embodiments, theadditional therapeutic agent is an anti-PD-1 antibody. In certainembodiments, the anti-PD-1 antibody is pembrolizumab. In certainembodiments, the anti-PD-1 antibody is nivolumab.

In certain embodiments, the additional therapeutic agent is an inhibitorof indoleamine-2,3-dioxygenase (IDO). In certain embodiments, theinhibitor is selected from the group consisting of epacadostat, F001287,indoximod, and NLG919. In certain embodiments, the inhibitor isepacadostat. In certain embodiments, the inhibitor is F001287. Incertain embodiments, the inhibitor is indoximod. In certain embodiments,the inhibitor is NLG919.

In certain embodiments, the additional therapeutic agent is a vaccine.In certain embodiments, the vaccine comprises a heat shock proteinpeptide complex (HSPPC) comprising a heat shock protein complexed withan antigenic peptide. In certain embodiments, the heat shock protein ishsc70 and is complexed with a tumor-associated antigenic peptide. Incertain embodiments, the heat shock protein is gp96 protein and iscomplexed with a tumor-associated antigenic peptide, wherein the HSPPCis derived from a tumor obtained from a subject. In certain embodiments,the additional therapeutic agent comprises a TCR. In certainembodiments, the additional therapeutic agent is a soluble TCR. Incertain embodiments, the additional therapeutic agent is a cellexpressing a TCR. In certain embodiments, the additional therapeuticagent is a cell expressing a chimeric antigen receptor. In certainembodiments, the additional therapeutic agent is an antibody thatspecifically binds to a peptide-MHC complex. In certain embodiments, theadditional therapeutic agent is an adjuvant. In one aspect, the presentinvention relates to (a) an antibody, polynucleotide, vector,recombinant host cell, and/or pharmaceutical composition of the presentinvention and (b) a vaccine for use as a medicament, for example, foruse in a method for the treatment of cancer, optionally wherein thevaccine comprises a heat shock protein peptide complex (HSPPC)comprising a heat shock protein complexed with an antigenic peptide. Inone aspect, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) a vaccine, optionallywherein the vaccine comprises a heat shock protein peptide complex(HSPPC) comprising a heat shock protein complexed with an antigenicpeptide.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of histograms showing the binding of anti-TIM-3antibodies pab2085 (IgG₁) and pab2088 (IgG₁) or an isotype controlantibody to wild type murine 1624-5 cells or 1624-5 cells engineered toexpress human TIM-3, as measured by flow cytometry.

FIGS. 2A and 2B are a pair of graphs showing the binding of anti-TIM-3antibodies pab2085 (IgG₁) (FIG. 2A) and pab2088 (IgG₁) (FIG. 2B) torecombinant human TIM-1 His (rhTIM-1 His), recombinant human TIM-4 His(rhTIM-4 His), recombinant human TIM-3 His (rhTIM-3 His), recombinanthuman TIM-3 Fc (rhTIM-3 Fc), and recombinant cynomolgus TIM-3 Fc(rcmTIM-3 Fc), as measured by a LUMINEX® microsphere assay. The medianfluorescence intensity (MFI) values are plotted against antibodyconcentrations.

FIGS. 3A, 3B, 3C, and 3D are a set of histograms showing the binding ofanti-TIM-3 antibodies or an isotype control antibody to murine 1624-5cells engineered to express human TIM-3 (FIGS. 3A and 3B) or cynomolgusTIM-3 (FIGS. 3C and 3D), as measured by flow cytometry. The anti-TIM-3antibodies tested in this study include pab2173, pab2174, pab2175,pab2176, pab2177, pab2178, pab2179, pab2180, pab2181, pab2182, pab2183,pab2184, pab2185, pab2186, pab2187, pab2188, pab2189, pab2190, pab2191,and pab2192, all of which contain an IgG₁ Fc region.

FIG. 4 is a graph showing the binding of anti-TIM-3 antibody pab2085,light-chain optimized variants (pab2184, pab2186, pab2187, pab2188,pab2189, pab2190, pab2191, and pab2192), or an isotype control antibodyto primary human CD8+ T cells activated by anti-CD3 and anti-CD28antibodies, measured by flow cytometry. The light chain optimizedvariants contain an IgG₁ variant Fc region. The MFI values are plottedagainst a series of antibody concentrations tested.

FIG. 5 is a graph showing the binding of the anti-TIM-3 antibody pab2188(IgG₁ variant) or an isotype control antibody to primary cynomolgusCD11b+ myeloid cells, measured by flow cytometry. The MFI values areplotted against antibody concentrations.

FIGS. 6A and 6B are graphs showing the percent of binding betweenirradiated phosphatidylserine-expressing WR19L murine lymphoma cells andrecombinant human TIM-3 Fc (FIG. 6A) or recombinant cynomolgus TIM-3 Fc(FIG. 6B) in the presence of a dose titration of an anti-TIM-3 antibodyor an IgG₁ isotype control antibody. The anti-TIM-3 antibodies tested inthis study are pab2085 (IgG₁) and pab2188 (IgG₁ variant).

FIG. 7 is a bar graph showing the production of IFNγ induced byanti-TIM-3 antibodies or an IgG₁ isotype control antibody in combinationwith the anti-PD-1 antibody pembrolizumab in human peripheral bloodmononuclear cells (PBMCs) upon Staphylococcus Enterotoxin A (SEA)stimulation. The anti-TIM-3 antibodies tested in this study include thelight-chain optimized variants pab2175 (Ig pab2176 (Ig pab2180 (Igpab2182 (IgG₁), pab2183 (IgG₁ variant), pab2184 (IgG₁ variant), pab2186(IgG₁ variant), pab2187 (IgG₁ variant), pab2188 (IgG₁ variant), pab2189(IgG₁ variant), pab2190 (IgG₁ variant), pab2191 (IgG₁ variant), andpab2192 (IgG₁ variant).

FIGS. 8A, 8B, 8C, 8D, 8E, and 8F are a set of bar graphs showing theproduction of IFNγ induced by the anti-TIM-3 antibody pab2188w (IgG₁N297A) or an IgG₁ N297A isotype control antibody, either alone or incombination with the anti-PD-1 antibody pembrolizumab, in human PBMCsfrom six different donors upon SEA stimulation. The protocol used in thestudy depicted in FIGS. 8A-8F was modified from the protocol used in thestudy depicted in FIG. 7.

FIGS. 9A, 9B, 9C, 9D, 9E, and 9F are graphs or histograms showingbinding of anti-TIM-3 antibodies to cells expressing TIM-3. In FIGS. 9A,9B, 9E, and 9F, MFI values are plotted against a series of antibodyconcentrations tested. FIGS. 9C and 9D are a set of histograms showingbinding of anti-TIM-3 antibodies to TIM-3-expressing cells. Theanti-TIM-3 antibodies tested include pab2188w (IgG₁ N297A), AM-1 (IgG₁N297A), AM-2 (IgG₁ N297A), AM-3 (IgG₁ N297A), AM-4 (IgG₁ N297A), AM-5(IgG₁ N297A), AM-6 (IgG₁ N297A), AM-7 (IgG₁ N297A), AM-8 (IgG₁ N297A),and AM-9 (IgG₁ N297A). The cells tested were Jurkat cells ectopicallyexpressing human TIM-3 (FIG. 9A), Kasumi-3, a human acute myeloidleukemia cell line endogenously expressing TIM-3 (FIG. 9B), human CD8+ Tcells stimulated with Staphylococcal Enterotoxin A (SEA) (FIG. 9C),cynomolgus CD8+ T cells stimulated with SEA (FIG. 9D), and primary human(FIG. 9E) and cynomolgus (FIG. 9F) CD14+ myeloid cells.

FIGS. 10A, 10B, 10C, and 10D are graphs showing the binding ofanti-TIM-3 antibodies or an IgG₁ N297A isotype control antibody torecombinant human TIM-3 His (rhTIM-3 His), recombinant cynomolgus TIM-3Fc (rcmTIM-3 Fc), recombinant mouse TIM-3 Fc (rmTIM-3 Fc), recombinanthuman TIM-1 His (rhTIM-1 His), recombinant human TIM-4 His (rhTIM-4His), recombinant human OX40 His (rhOX40 His), recombinant human GITR Fc(rhGITR Fc), recombinant human DR3 Fc (rhDR3 Fc), and recombinant humanCD137 Fc (rhCD137 Fc), measured by a LUMINEX® microsphere assay. The MFIvalues are plotted against antibody concentrations. The anti-TIM-3antibodies tested in this study include pab2188w (IgG₁ N297A) (FIG.10B), AM-2 (IgG₁ N297A) (FIG. 10C), and AM-6 (IgG₁ N297A) (FIG. 10D).

FIGS. 11A and 11B are graphs showing the percent of binding ofrecombinant human TIM-3 Fc (FIG. 11A) or recombinant cynomolgus TIM-3 Fc(FIG. 11B) to phosphatidylserine-expressing WR19L cells in the presenceof a dose titration of anti-TIM-3 antibodies or an IgG₁ N297A isotypecontrol antibody. The anti-TIM-3 antibodies tested in this study includepab2188w (IgG₁ N297A), AM-2 (IgG₁ N297A), and AM-6 (IgG₁ N297A).

FIGS. 12A and 12B are bar graphs showing the production of IFNγ inducedby anti-TIM-3 antibodies or an IgG₁ N297A isotype control antibody,either alone or in combination with the anti-PD-1 antibodypembrolizumab, in human PBMCs from two different donors upon SEAstimulation. The anti-TIM-3 antibodies tested include pab2188w (IgG₁N297A), AM-1 (IgG₁ N297A), AM-2 (IgG₁ N297A), AM-3 (IgG₁ N297A), AM-4(IgG₁ N297A), AM-5 (IgG₁ N297A), AM-6 (IgG₁ N297A), AM-7 (IgG₁ N297A),and AM-8 (IgG₁ N297A).

FIGS. 13A, 13B, 13C, 13D, 13E, and 13F are graphs showing IFNγ or TNFαproduction by primary tumor infiltrating lymphocytes (TILs) induced byanti-TIM-3 antibodies or an IgG₁ N297A isotype control antibody, eitheralone or in combination with the anti-PD-1 antibody pembrolizumab. TheTILs were isolated from non-small cell lung cancer (NSCLC) (FIGS. 13Aand 13B), gallbladder adenocarcinoma (FIGS. 13C and 13D), or breastcancer (FIGS. 13E and 13F) tumors and activated with anti-CD3/CD28microbeads. The anti-TIM-3 antibodies tested in this study includepab2188w (IgG₁ N297A), AM-2 (IgG₁ N297A), and AM-6 (IgG₁ N297A).

FIGS. 14A, 14B, and 14C are graphs showing the percent of cell survival,relative to an untreated control group, after incubation with ananti-TIM-3 antibody or an IgG₁ N297A isotype control antibody. FIGS. 14Aand 14B show treatment with the indicated antibody in combination with asecondary antibody drug conjugate αHFc-NC-DM1. The cells tested wereJurkat cells engineered to overexpress TIM-3 (FIG. 14A) or Kasumi-3cells, an acute myeloid leukemia cell line endogenously expressing TIM-3(FIG. 14B). FIG. 14C shows treatment with the indicated antibody as aconjugate with monomethyl auristatin E (MMAE). The anti-TIM-3 antibodiestested in this study include pab2188w (IgG₁ N297A), AM-2 (IgG₁ N297A),AM-6 (IgG₁ N297A), and reference antibodies Hum11 (IgG₄ S228P) andpab1944w (IgG₁ N297A).

FIG. 15 is a series of graphs showing TIM-3 internalization in Jurkatcells expressing a HALOTAG® peptide tag-TIM-3 fusion protein whenincubated with 10 μg/mL of either anti-TIM-3 antibody AM-2 or an isotypecontrol antibody, as determined by live cell confocal fluorescencemicroscopy, at various time points (i.e., at 0-3.5 hours). Black dotsindicate the mean fluorescence level observed for each condition at agiven time point.

5. DETAILED DESCRIPTION

The instant disclosure provides antibodies that specifically bind toTIM-3 (e.g., human TIM-3) and antagonize TIM-3 function, e.g.,TIM-3-mediated immune suppression. Also provided are pharmaceuticalcompositions comprising these antibodies, nucleic acids encoding theseantibodies, expression vectors and host cells for making theseantibodies, and methods of treating a subject using these antibodies.The antibodies disclosed herein are particularly useful for increasing Tcell activation in response to an antigen (e.g., a tumor antigen or aninfectious disease antigen), and hence for treating cancer in a subjector treating or preventing an infectious disease in a subject. Allinstances of “isolated antibodies” described herein are additionallycontemplated as antibodies that may be, but need not be, isolated. Allinstances of “isolated polynucleotides” described herein areadditionally contemplated as polynucleotides that may be, but need notbe, isolated. All instances of “antibodies” described herein areadditionally contemplated as antibodies that may be, but need not be,isolated. All instances of “polynucleotides” described herein areadditionally contemplated as polynucleotides that may be, but need notbe, isolated.

5.1 Definitions

As used herein, the terms “about” and “approximately,” when used tomodify a numeric value or numeric range, indicate that deviations of 5%to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., upto 5% to 10% below) the value or range remain within the intendedmeaning of the recited value or range.

As used herein, the term “TIM-3” refers to T cell immunoglobulin andmucin domain-3 (also known as T cell immunoglobulin and mucin-domaincontaining-3 protein or Hepatitis A virus cellular receptor 2 (HAVCR2))that in humans is encoded by the HAVCR2 gene. Swiss-Prot accessionnumber Q8TDQ0-1 provides an exemplary human TIM-3 amino acid sequence.The immature amino acid sequence of human TIM-3 is provided as SEQ IDNO: 78. The mature amino acid sequence of human TIM-3 is provided as SEQID NO: 79. As used herein, the term “human TIM-3” refers to TIM-3comprising the amino acid sequence of SEQ ID NO: 79.

As used herein, the terms “antibody” and “antibodies” include fulllength antibodies, antigen-binding fragments of full length antibodies,and molecules comprising antibody CDRs, VH regions or VL regions.Examples of antibodies include monoclonal antibodies, recombinantlyproduced antibodies, monospecific antibodies, multispecific antibodies(including bispecific antibodies), human antibodies, humanizedantibodies, chimeric antibodies, immunoglobulins, synthetic antibodies,tetrameric antibodies comprising two heavy chain and two light chainmolecules, an antibody light chain monomer, an antibody heavy chainmonomer, an antibody light chain dimer, an antibody heavy chain dimer,an antibody light chain-antibody heavy chain pair, intrabodies,heteroconjugate antibodies, antibody-drug conjugates, single domainantibodies, monovalent antibodies, single chain antibodies orsingle-chain Fvs (scFv), camelized antibodies, affybodies, Fabfragments, F(ab′)2 fragments, disulfide-linked Fvs (sdFv),anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Idantibodies), and antigen-binding fragments of any of the above. Incertain embodiments, antibodies described herein refer to polyclonalantibody populations. Antibodies can be of any type (e.g., IgG, IgE,IgM, IgD, IgA or IgY), any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ orIgA₂), or any subclass (e.g., IgG_(2a) or IgG_(2b)) of immunoglobulinmolecule. In certain embodiments, antibodies described herein are IgGantibodies, or a class (e.g., human IgG₁ or IgG₄) or subclass thereof.In a specific embodiment, the antibody is a humanized monoclonalantibody. In another specific embodiment, the antibody is a humanmonoclonal antibody.

As used herein, the terms “VH region” and “VL region” refer to singleantibody heavy and light chain variable regions, respectively,comprising FR (Framework Regions) 1, 2, 3 and 4 and CDR (ComplementarityDetermining Regions) 1, 2 and 3 (see Kabat et al., (1991) Sequences ofProteins of Immunological Interest (NIH Publication No. 91-3242,Bethesda), which is herein incorporated by reference in its entirety).

As used herein, the term “CDR” or “complementarity determining region”means the noncontiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), by Chothia et al., J. Mol. Biol.196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745(1996), all of which are herein incorporated by reference in theirentireties, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. In certainembodiments, the term “CDR” is a CDR as defined by MacCallum et al., J.Mol. Biol. 262:732-745 (1996) and Martin A. “Protein Sequence andStructure Analysis of Antibody Variable Domains,” in AntibodyEngineering, Kontermann and Dilbel, eds., Chapter 31, pp. 422-439,Springer-Verlag, Berlin (2001). In certain embodiments, the term “CDR”is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616(1977) and Kabat et al., Sequences of protein of immunological interest.(1991). In certain embodiments, heavy chain CDRs and light chain CDRs ofan antibody are defined using different conventions. For example, incertain embodiments, the heavy chain CDRs are defined according toMacCallum (supra), and the light CDRs are defined according to Kabat(supra). CDRH1, CDRH2 and CDRH3 denote the heavy chain CDRs, and CDRL1,CDRL2 and CDRL3 denote the light chain CDRs.

As used herein, the term “framework (FR) amino acid residues” refers tothose amino acids in the framework region of an immunoglobulin chain.The term “framework region” or “FR region” as used herein, includes theamino acid residues that are part of the variable region, but are notpart of the CDRs (e.g., using the Kabat or MacCallum definition ofCDRs).

As used herein, the terms “variable region” and “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, typically about the amino-terminal 110 to 120amino acids or 110 to 125 amino acids in the mature heavy chain andabout 90 to 115 amino acids in the mature light chain, which differextensively in sequence among antibodies and are used in the binding andspecificity of a particular antibody for its particular antigen. Thevariability in sequence is concentrated in those regions calledcomplementarity determining regions (CDRs) while the more highlyconserved regions in the variable domain are called framework regions(FR). Without wishing to be bound by any particular mechanism or theory,it is believed that the CDRs of the light and heavy chains are primarilyresponsible for the interaction and specificity of the antibody withantigen. In certain embodiments, the variable region is a human variableregion. In certain embodiments, the variable region comprises rodent ormurine CDRs and human framework regions (FRs). In particularembodiments, the variable region is a primate (e.g., non-human primate)variable region. In certain embodiments, the variable region comprisesrodent or murine CDRs and primate (e.g., non-human primate) frameworkregions (FRs).

The terms “VL” and “VL domain” are used interchangeably to refer to thelight chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to theheavy chain variable region of an antibody.

As used herein, the terms “constant region” and “constant domain” areinterchangeable and are common in the art. The constant region is anantibody portion, e.g., a carboxyl terminal portion of a light and/orheavy chain which is not directly involved in binding of an antibody toantigen but which can exhibit various effector functions, such asinteraction with an Fc receptor (e.g., Fc gamma receptor). The constantregion of an immunoglobulin molecule generally has a more conservedamino acid sequence relative to an immunoglobulin variable domain.

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG, and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃, and IgG₄.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are well known in the art. In specific embodiments,the light chain is a human light chain.

As used herein, the term “EU numbering system” refers to the EUnumbering convention for the constant regions of an antibody, asdescribed in Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85(1969) and Kabat et al, Sequences of Proteins of Immunological Interest,U.S. Dept. Health and Human Services, 5th edition, 1991, each of whichis herein incorporated by reference in its entirety.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured and/or expressedin a number of ways known in the art, including, but not limited to,equilibrium dissociation constant (K_(D)), and equilibrium associationconstant (K_(A)). The K_(D) is calculated from the quotient ofk_(off)/k_(on), whereas K_(A) is calculated from the quotient ofk_(on)/k_(off). k_(on) refers to the association rate constant of, e.g.,an antibody to an antigen, and k_(off) refers to the dissociation rateconstant of, e.g., an antibody to an antigen. The k_(on) and k_(off) canbe determined by techniques known to one of ordinary skill in the art,such as a BIACORE™ surface plasmon resonance assay or a KINEXA® (kineticexclusion assay). As used herein, a “lower affinity” refers to a largerK_(D).

As used herein, the terms “specifically binds,” “specificallyrecognizes,” “immunospecifically binds,” and “immunospecificallyrecognizes” are analogous terms in the context of antibodies and referto molecules that bind to an antigen (e.g., epitope or immune complex)as such binding is understood by one skilled in the art. For example, amolecule that specifically binds to an antigen can bind to otherpeptides or polypeptides, generally with lower affinity as determinedby, e.g., immunoassays, BIACORE™ surface plasmon resonance assay,KINEXA® 3000 instrument (Sapidyne Instruments, Boise, Id.), or otherassays known in the art. In a specific embodiment, molecules thatspecifically bind to an antigen bind to the antigen with a K_(A) that isat least 2 logs (e.g., factors of 10), 2.5 logs, 3 logs, 4 logs orgreater than the K_(A) when the molecules bind non-specifically toanother antigen.

In another specific embodiment, molecules that specifically bind to anantigen do not cross react with other proteins under similar bindingconditions. In another specific embodiment, molecules that specificallybind to TIM-3 do not cross react with other non-TIM-3 proteins. In aspecific embodiment, provided herein is an antibody that binds to TIM-3(e.g., human TIM-3) with higher affinity than to another unrelatedantigen. In certain embodiments, provided herein is an antibody thatbinds to TIM-3 (e.g., human TIM-3) with a 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher affinity thanto another, unrelated antigen as measured by, e.g., a radioimmunoassay,surface plasmon resonance, or kinetic exclusion assay. In a specificembodiment, the extent of binding of an anti-TIM-3 antibody describedherein to an unrelated, non-TIM-3 protein is less than 10%, 15%, or 20%of the binding of the antibody to TIM-3 protein as measured by, e.g., aradioimmunoassay.

As used herein, the term “afucosylation” or “afucosylated” in thecontext of an Fc refers to a substantial lack of a fucose covalentlyattached, directly or indirectly, to residue 297 of the human IgG₁ Fcregion, numbered according to the EU numbering system, or thecorresponding residue in non-IgG₁ or non-human IgG₁ immunoglobulins.Thus, in a composition comprising a plurality of afucosylatedantibodies, at least 70% of the antibodies will not be fucosylated,directly or indirectly (e.g., via intervening sugars) at residue 297 ofthe Fc region of the antibodies, and in some embodiments at least 80%,85%, 90%, 95%, or 99% will not be fucosylated, directly or indirectly,at residue 297 of the Fc region.

As used herein, an “epitope” is a term in the art and refers to alocalized region of an antigen to which an antibody can specificallybind. An epitope can be, for example, contiguous amino acids of apolypeptide (linear or contiguous epitope) or an epitope can, forexample, come together from two or more non-contiguous regions of apolypeptide or polypeptides (conformational, non-linear, discontinuous,or non-contiguous epitope). In certain embodiments, the epitope to whichan antibody binds can be determined by, e.g., NMR spectroscopy, X-raydiffraction crystallography studies, ELISA assays, hydrogen/deuteriumexchange coupled with mass spectrometry (e.g., liquid chromatographyelectrospray mass spectrometry), array-based oligo-peptide scanningassays (e.g., constraining peptides using CLIPS (Chemical Linkage ofPeptides onto Scaffolds) to map discontinuous or conformationalepitopes), and/or mutagenesis mapping (e.g., site-directed mutagenesismapping). For X-ray crystallography, crystallization may be accomplishedusing any of the known methods in the art (e.g., Giegé R et al., (1994)Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A(1990) Eur J Biochem 189: 1-23; Chayen N E (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, each of whichis herein incorporated by reference in its entirety). Antibody:antigencrystals may be studied using well known X-ray diffraction techniquesand may be refined using computer software such as X-PLOR (YaleUniversity, 1992, distributed by Molecular Simulations, Inc.; see, e.g.,Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H W et al.; U.S.2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D BiolCrystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D BiolCrystallogr 56(Pt 10): 1316-1323), each of which is herein incorporatedby reference in its entirety. Mutagenesis mapping studies may beaccomplished using any method known to one of skill in the art. See,e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and CunninghamB C & Wells J A (1989) Science 244: 1081-1085, each of which is hereinincorporated by reference in its entirety, for a description ofmutagenesis techniques, including alanine scanning mutagenesistechniques. CLIPS (Chemical Linkage of Peptides onto Scaffolds) is atechnology to present one or more peptides in a structurally constrainedconfiguration to behave as functional mimics of complex protein domains.See, e.g., U.S. Publication Nos. US 2008/0139407 A1 and US 2007/099240A1, and U.S. Pat. No. 7,972,993, each of which is herein incorporated byreference in its entirety. In a specific embodiment, the epitope of anantibody is determined using alanine scanning mutagenesis studies. In aspecific embodiment, the epitope of an antibody is determined usinghydrogen/deuterium exchange coupled with mass spectrometry. In aspecific embodiment, the epitope of an antibody is determined usingCLIPS Epitope Mapping Technology from Pepscan Therapeutics.

As used herein, the term “an epitope located within a region of humanTIM-3” consisting of a particular amino acid sequence or a set of aminoacid residues refers to an epitope comprising one or more of the aminoacid residues of the specified region, wherein the specified regionincludes the first specified amino acid residue and the last specifiedamino acid residue of the region of human TIM-3. In certain embodiments,the epitope comprises each one of the amino acid residues located withinthe specified region. In certain embodiments, one or more additionalamino acid residues of human TIM-3 outside the specified region bind toan antibody together with an epitope located within the specifiedregion.

As used herein, the terms “T cell receptor” and “TCR” are usedinterchangeably and refer to full length heterodimeric αβ or γδ TCRs,antigen-binding fragments of full length TCRs, and molecules comprisingTCR CDRs or variable regions. Examples of TCRs include, but are notlimited to, full length TCRs, antigen-binding fragments of full lengthTCRs, soluble TCRs lacking transmembrane and cytoplasmic regions,single-chain TCRs containing variable regions of TCRs attached by aflexible linker, TCR chains linked by an engineered disulfide bond,monospecific TCRs, multi-specific TCRs (including bispecific TCRs), TCRfusions, human TCRs, humanized TCRs, chimeric TCRs, recombinantlyproduced TCRs, and synthetic TCRs. The term encompasses wild-type TCRsand genetically engineered TCRs (e.g., a chimeric TCR comprising achimeric TCR chain which includes a first portion from a TCR of a firstspecies and a second portion from a TCR of a second species).

As used herein, the terms “major histocompatibility complex” and “MHC”are used interchangeably and refer to an MHC class I molecule and/or anMHC class II molecule.

As used herein, the term “peptide-MHC complex” refers to an MHC molecule(MHC class I or MHC class II) with a peptide bound in the art-recognizedpeptide binding pocket of the MHC.

As used herein, the term “treat,” “treating,” and “treatment” refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration of an antibody to a subject having adisease or disorder, or predisposed to having such a disease ordisorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disease or disorder or recurringdisease or disorder, or in order to prolong the survival of a subjectbeyond that expected in the absence of such treatment.

As used herein, the term “effective amount” in the context of theadministration of a therapy to a subject refers to the amount of atherapy that achieves a desired prophylactic or therapeutic effect.

As used herein, the term “subject” includes any human or non-humananimal. In one embodiment, the subject is a human or non-human mammal.In one embodiment, the subject is a human.

The determination of “percent identity” between two sequences (e.g.,amino acid sequences or nucleic acid sequences) can be accomplishedusing a mathematical algorithm. A specific, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin S & Altschul S F (1990) PNAS 87: 2264-2268,modified as in Karlin S & Altschul S F (1993) PNAS 90: 5873-5877, eachof which is herein incorporated by reference in its entirety. Such analgorithm is incorporated into the NBLAST and)(BLAST programs ofAltschul S F et al., (1990) J Mol Biol 215: 403, which is hereinincorporated by reference in its entirety. BLAST® (basic local alignmentsearch tool) nucleotide searches can be performed with the NBLASTnucleotide program parameters set, e.g., for score=100, wordlength=12 toobtain nucleotide sequences homologous to a nucleic acid moleculesdescribed herein. BLAST® protein searches can be performed withthe)(BLAST program parameters set, e.g., to score 50, wordlength=3 toobtain amino acid sequences homologous to a protein molecule describedherein. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul S F et al., (1997) NucAcids Res 25: 3389-3402, which is herein incorporated by reference inits entirety. Alternatively, PSI BLAST can be used to perform aniterated search which detects distant relationships between molecules(Id.). When utilizing BLAST®, Gapped BLAST, and PSI Blast programs, thedefault parameters of the respective programs (e.g., of)(BLAST andNBLAST) can be used (see, e.g., National Center for BiotechnologyInformation (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Anotherspecific, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17, which is herein incorporated by reference in itsentirety. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

As used herein, the term “internalization” or “internalized” refers tothe uptake of an antibody into an intracellular compartment of a cellupon binding of the antibody to an antigen expressed at the surface ofthe cell.

5.2 Anti-TIM-3 Antibodies

In one aspect, the instant disclosure provides antibodies thatspecifically bind to TIM-3 (e.g., human TIM-3) and antagonize TIM-3function. The amino acid sequences of exemplary antibodies are set forthin Tables 1-4, herein.

TABLE 1 Amino acid sequences of exemplary anti-TIM-3 antibodies. SEQID NO: Description* Amino acid sequence 1 BADD456-2919 CDRH1 SSYAMS 2BADD456-2919 CDRH2 WVSAISGSGGSTY 3 BADD456-2919 CDRH3 AKGGDYGGNYFD 4AM-1 CDRH1 KAGQSS 5 AM-2 CDRH1 RQNAWS 6 AM-3 CDRH1 MSGQTS 7 AM-4 CDRH1GAGQSS 8 AM-5 CDRH1 SAQQAS 9 AM-6 CDRH1 ARNAWS 10 AM-7 CDRH1 RSQQAS 11AM-8 CDRH1 TTQQAS 12 AM-9 CDRH1 GGQQAS 13 BADD197-1181 CDRL1RASQSVSSSYLA 14 BADD412-2513 CDRL1 RASQSVSSYLA 15 BADD456-2928 CDRL1RASQGISNYLA 16 BADD466-3169 CDRL1 GASQSVSSSYLA 17 BADD197-1181 CDRL2GASSRAT 18 BADD456-2928 CDRL2 AASTLQS 19 BADD466-3165 CDRL2 GASTRAT 20BADD466-3166 CDRL2 DASSRAT 21 BADD466-3167 CDRL2 DASNRAT 22BADD197-1181 CDRL3 QQYGSSPLT 23 BADD392-2234 CDRL3 QQYGSSPIT 24BADD456-2919 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 25 BADD466-3162 VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAM SWVRRAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGD YGGNYFDYWGQGTLVTVSS 26BADD466-3163 VH EVQLVESGGGLVQPRGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 27 AM-1-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFKAGQSSWVRRAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 28 AM-2-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFRQNAWSWVRRAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 29 AM-3 -VH EVQLVESGGGLVQPGGSLRLSCAASGFTFMSGQTSWVRRAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 30 AM-4-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFGAGQSSWVRRAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 31 AM-5-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSAQQASWVRRAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 32 AM-6-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFARNAWSWVRRAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 33 AM-7-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFRSQQASWVRRAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 34 AM-8-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTTQQASWVRRAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 35 AM-9-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFGGQQASWVRRAPGKGLEWVSAISGSGGSTYYADSVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS 36 BADD197-1181 VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLA WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKV EIK 37 BADD412-2513 VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEI K 38 BADD456-2928 VLDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAW YQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEI K 39 BADD466-3164 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVK IK 40 BADD466-3165 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEI K 41 BADD466-3166 VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEI K 42 BADD466-3167 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEI K 43 BADD466-3168 VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEI K 44 BADD466-3169 VLEIVLTQSPATLSLSPGERATLSCGASQSVSSSYLA WYQQKPGLAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKV EIK 45 BADD466-3170 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSPLTFGGGTKVEI K 46 BADD466-3171 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDASNRATGIPASFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEI K 47 BADD466-3172 VLEIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYGASSRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGGGTKVE IK 48 CDRH1 consensusX₁X₂X₃X₄X₅S, wherein: sequence 1 X₁ is R, S, A, G, K, M, or T;X₂ is Q, S, A, G, R, or T; X₃ is N, Y, G, or Q; X₄ is A or Q; andX₅ is W, M, A, S, or T 49 CDRH1 consensus X₁X₂NAWS, wherein: sequence 2X₁ is R or A; and X₂ is Q or R 50 CDRH1 consensus X₁X₂GQX₃S, wherein:sequence 3 X₁ is K, M, or G; X₂ is A or S; and X₃ is S or T 51CDRH1 consensus X₁X₂QQAS, wherein: sequence 4 X₁ is S, R, T, or G; andX₂ is A, S, T, or G 52 CDRL1 consensus X₁ASQSVX₂SSYLA, wherein sequenceX₁ is R or G; and X₂ is absent or S 53 CDRL2 consensusX₁ASX₂RAT, wherein: sequence X₁ is D or G; and X₂is N, S, or T 54CDRL3 consensus QQYGSSPX₁T, wherein sequence X₁ is L or I 55VH consensus EVQLVESGGGLVQPX₁GSLRLSCAASGFTF sequenceX₂X₃X₄X₅X₆SWVRX₇APGKGLEWVSAISG SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQG TLVTVSS, wherein: X₁ is G or R;X₂ is R, S, A, G, K, M, or T; X₃ is Q, S, A, G, R, or T;X₄ is N, Y, G, or Q; X₅ is A or Q; X₆ is W, M, A, S, or T; andX₇ is R or Q 56 VL consensus EIVLTQSPX₁TLSLSPGERATLSCX₂ASQSVX₁SSYLsequence AWYQQKPGX₄APRLLIYX₅ASX₆RATGIPX₇X₈FSGSGSGTDFTLTISX₉LEPEDFAVYYCQQYGSSPX₁₀TFG GGTKVX₁₁IK, wherein: X₁ is A or G;X₂ is R or G; X₃ is absent or S; X₄ is O or L; X₅ is D or G;X₆ is N, S, or T; X₇ is A or D; X₈ is S or R; X₉ is R or S;X₁₀ is L or I; and X₁₁ is E or K 57 pab2188 fullEVQLVESGGGLVQPGGSLRLSCAAS length IgG₁ GFTFSSYAMSWVRRAPGKGLEWVSAheavy chain ISGSGGSTYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG 58 pab2188 fullEVQLVESGGGLVQPGGSLRLSCAAS length IgG₁ GFTFSSYAMSWVRRAPGKGLEWVSA N297AISGSGGSTYYADSVKGRFTISRDNS heavy chain KNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGQLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPLEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG 59 pab2188 fullEVQLVESGGGLVQPGGSLRLSCAAS length IgG₄ GFTFSSYAMSWVRRAPGKGLEWVSAS228P heavy ISGSGGSTYYADSVKGRFTISRDNS chain KNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLG 60 AM-1 fullEVQLVESGGGLVQPGGSLRLSCAAS length IgG₁ GFTFKAGQSSWVRRAPGKGLEWVSAN297A heavy ISGSGGSTYYADSVKGRFTISRDNS chain KNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG 61 AM-2 fullEVQLVESGGGLVQPGGSLRLSCAAS length IgG₁ GFTFRQNAWSWVRRAPGKGLEWVSAN297A heavy ISGSGGSTYYADSVKGRFTISRDNS chain KNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGQLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG 62 AM-3 fullEVQLVESGGGLVQPGGSLRLSCAASG length IgG₁ FTFMSGQTSWVRRAPGKGLEWVSAISN297A heavy GSGGSTYYADSVKGRFTISRDNSKNT chain LYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 63 AM-4 fullEVQLVESGGGLVQPGGSLRLSCAASG length IgG₁ FTFGAGQSSWVRRAPGKGLEWVSAISN297A heavy GSGGSTYYADSVKGRFTISRDNSKNT chain LYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 64 AM-5 fullEVQLVESGGGLVQPGGSLRLSCAASG length IgG₁ FTFSAQQASWVRRAPGKGLEWVSAISN297A heavy GSGGSTYYADSVKGRFTISRDNSKNT chain LYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGQLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 65 AM-6 fullEVQLVESGGGLVQPGGSLRLSCAASG length IgG₁ FTFARNAWSWVRRAPGKGLEWVSAISN297A heavy GSGGSTYYADSVKGRFTISRDNSKNT chain LYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 66 AM-7 fullEVQLVESGGGLVQPGGSLRLSCAAS length IgG₁ GFTFRSQQASWVRRAPGKGLEWVSAN297A heavy ISGSGGSTYYADSVKGRFTISRDNS chain KNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG 67 AM-8 fullEVQLVESGGGLVQPGGSLRLSCAA length IgG₁ SGFTFTTQQASWVRRAPGKGLEWVN297A heavy SAISGSGGSTYYADSVKGRFTISR chain DNSKNTLYLQMNSLRAEDTAVYYCAKGGDYGGNYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG 68AM-9 full EVQLVESGGGLVQPGGSLRLSCAAS length IgG₁GFTFGGQQASWVRRAPGKGLEWVSA N297A heavy ISGSGGSTYYADSVKGRFTISRDNS chainKNTLYLQMNSLRAEDTAVYYCAKGG DYGGNYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 69 BADD466-3171 EIVLTQSPATLSLSPGERATLSCRAfull length SQSVSSYLAWYQQKPGQAPRLLIYD light chainASNRATGIPASFSGSGSGTDFTLTI sequence SRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 70Human IgG₁ ASTKGPSVFPLAPSSKSTSGGTAAL G1m3 GCLVKDYFPEPVTVSWNSGALTSGVallotype HTFPAVLQSSGLYSLSSVVTVPSSS (without C- LGTQTYICNVNHKPSNTKVDKRVEPterminal KSCDKTHTCPPCPAPELLGGPSVFL lysine) FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 71 Human IgG₁ ASTKGPSVFPLAPSSKSTSGGTAALG1m3 GCLVKDYFPEPVTVSWNSGALTSGV allotype HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLS LSPGK 72 IgG₁ N297AASTKGPSVFPLAPSSKSTSGGTAALG (without C- CLVKDYFPEPVTVSWNSGALTSGVHTterminal FPAVLQSSGLYSLSSWTVPSSSLGTQ lysine) TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG73 IgG₁ N297A ASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK74 IgG₄ S228P ASTKGPSVFPLAPCSRSTSESTAALG (without C-CLVKDYFPEPVTVSWNSGALTSGVHT terminal FPAVLQSSGLYSLSSVVTVPSSSLGT lysine)KTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLG 75 IgG₄ S228PASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 76 Human kappa RTVAAPSVFIFPPSDEQLKSGTASVlight chain VCLLNNFYPREAKVQWKVDNALQSG constant NSQESVTEQDSKDSTYSLSSTLTLSregion IGKC*01 KADYEKHKVYACEVTHQGLSSPVTK Km3 allotype SFNRGEC 77Human kappa RSVAAPSVFIFPPSDEQLKSGTASV light chainVCLLNNFYPREAKVQWKVDNALQSG constant NSQESVTEQDSKDSTYSLSSTLTLSregion IGKC*01 KADYEKHKVYACEVTHQGLSSPVTK Km3 allotype SFNRGEC (withT109S mutation) 84 IGHV3-23*04 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAIS GSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 85 IGKV1-27*01 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAAS TLQSGVPSRFSGSGSGTDFTLTISSL QPEDVATYYCQKYNSAP86 IGKV3-11*01 EIVLTQSPATLSLSPGERATLSCRAS QSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWP 87 IGKV3-20*01EIVLTQSPGTLSLSPGERATLSCRAS QSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSSP 88 IGKV3D-20*01EIVLTQSPATLSLSPGERATLSCGAS QSVSSSYLAWYQQKPGLAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSSP *Heavy chain CDRs aredefined according to the MacCallum numbering system and light chain CDRsare defined according to the Kabat numbering system.

TABLE 2 Heavy chain CDR amino acid sequencesof exemplary anti-TIM-3 antibodies. SEQ SEQ SEQ ID ID ID VH CDRH1* NO:CDRH2* NO: CDRH3* NO: BADD456- SSYAMS 1 WVSAISG 2 AKGGDY 3 2919 SGGSTYGGNYFD BADD466- SSYAMS 1 WVSAISG 2 AKGGDY 3 3162 SGGSTY GGNYFD BADD466-SSYAMS 1 WVSAISG 2 AKGGDY 3 3163 SGGSTY GGNYFD +0AM-1 KAGQSS 4 WVSAISG 2AKGGDY 3 SGGSTY GGNYFD +0AM-2 RQNAWS 5 WVSAISG 2 AKGGDY 3 SGGSTY GGNYFD+0AM-3 MSGQTS 6 WVSAISG 2 AKGGDY 3 SGGSTY GGNYFD +0AM-4 GAGQSS 7 WVSAISG2 AKGGDY 3 SGGSTY GGNYFD +0AM-5 SAQQAS 8 WVSAISG 2 AKGGDY 3 SGGSTYGGNYFD +0AM-6 ARNAWS 9 WVSAISG 2 AKGGDY 3 SGGSTY GGNYFD +0AM-7 RSQQAS 10WVSAISG 2 AKGGDY 3 SGGSTY GGNYFD +0AM-8 TTQQAS 11 WVSAISG 2 AKGGDY 3SGGSTY GGNYFD +0AM-9 GGQQAS 12 WVSAISG 2 AKGGDY 3 SGGSTY GGNYFD *Definedaccording to the MacCallum numbering system.

TABLE 3 Light chain CDR amino acid sequencesof exemplary anti-TIM-3 antibodies. SEQ SEQ SEQ ID ID ID VL CDRL1* NO:CDRL2* NO: CDRL3* NO: BADD197- RASQSV 13 GASSRAT 17 QQYGS 22 1181 SSSYLASPLT BADD412- RASQSV 14 GASSRAT 17 QQYGS 22 2513 SSYLA SPLT BADD456-RASQGI 15 AASTLQS 18 QQYGS 22 2928 SNYLA SPLT BADD466- RASQSV 14 GASSRAT17 QQYGS 22 3164 SSYLA SPLT BADD466- RASQSV 14 GASTRAT 19 QQYGS 22 3165SSYLA SPLT BADD466- RASQSV 14 DASSRAT 20 QQYGS 22 3166 SSYLA SPLTBADD466- RASQSV 14 DASNRAT 21 QQYGS 22 3167 SSYLA SPLT BADD466- RASQSV14 DASNRAT 21 QQYGS 22 3168 SSYLA SPLT BADD466- GASQSV 16 DASSRAT 20QQYGS 22 3169 SSSYLA SPLT BADD466- RASQSV 14 DASNRAT 21 QQYGS 22 3170SSYLA SPLT BADD466- RASQSV 14 DASNRAT 21 QQYGS 22 3171 SSYLA SPLTBADD466- RASQSV 14 GASSRAT 17 QQYGS 23 3172 SSYLA SPIT *Definedaccording to the Kabat numbering system.

TABLE 4 Exemplary anti-TIM-3 antibodies. Heavy chain SEQ ID Light chainSEQ ID Antibody variable region NO: variable region NO: pab2085BADD456-2919 24 BADD197-1181 36 pab2088 BADD456-2919 24 BADD456-2928 38pab2173 BADD466-3163 26 BADD466-3167 42 pab2174 BADD456-2919 24BADD466-3167 42 pab2175 BADD456-2919 24 BADD466-3171 46 pab2176BADD456-2919 24 BADD466-3168 43 pab2177 BADD466-3163 26 BADD466-3168 43pab2178 BADD466-3163 26 BADD466-3171 46 pab2179 BADD466-3163 26BADD466-3166 41 pab2180 BADD456-2919 24 BADD466-3166 41 pab2181BADD466-3162 25 BADD466-3164 39 pab2182 BADD456-2919 24 BADD466-3172 47pab2183 BADD466-3162 25 BADD466-3165 40 pab2184 BADD466-3163 26BADD466-3172 47 pab2185 BADD466-3162 25 BADD412-2513 37 pab2186BADD466-3162 25 BADD466-3170 45 pab2187 BADD466-3162 25 BADD466-3169 44pab2188 BADD466-3162 25 BADD466-3171 46 pab2189 BADD466-3162 25BADD466-3167 42 pab2190 BADD466-3162 25 BADD466-3166 41 pab2191BADD466-3162 25 BADD466-3168 43 pab2192 BADD466-3162 25 BADD466-3172 47AM-1 AM-1-VH 27 BADD466-3171 46 AM-2 AM-2-VH 28 BADD466-3171 46 AM-3AM-3-VH 29 BADD466-3171 46 AM-4 AM-4-VH 30 BADD466-3171 46 AM-5 AM-5-VH31 BADD466-3171 46 AM-6 AM-6-VH 32 BADD466-3171 46 AM-7 AM-7-VH 33BADD466-3171 46 AM-8 AM-8-VH 34 BADD466-3171 46 AM-9 AM-9-VH 35BADD466-3171 46

TABLE 5 Closest germline genes. Heavy chain or light chain Closestgermline SEQ ID NO for variable region gene germline gene: BADD456-2919VH IGHV3-23*04 84 BADD466-3162 VH IGHV3-23*04 84 BADD466-3163 VHIGHV3-23*04 84 AM-1-VH IGHV3-23*04 84 AM-2-VH IGHV3-23*04 84 AM-3-VHIGHV3-23*04 84 AM-4-VH IGHV3-23*04 84 AM-5-VH IGHV3-23*04 84 AM-6-VHIGHV3-23*04 84 AM-7-VH IGHV3-23*04 84 AM-8-VH IGHV3-23*04 84 AM-9-VHIGHV3-23*04 84 BADD197-1181 VL IGKV3-20*01 87 BADD412-2513 VLIGKV3-20*01 87 BADD456-2928 VL IGKV1-27*01 85 BADD466-3164 VLIGKV3-20*01 87 BADD466-3165 VL IGKV3-20*01 87 BADD466-3166 VLIGKV3-20*01 87 BADD466-3167 VL IGKV3-11*01 86 BADD466-3168 VLIGKV3-20*01 87 BADD466-3169 VL IGKV3D-20*01 88 BADD466-3170 VLIGKV3-11*01 86 BADD466-3171 VL IGKV3-11*01 86 BADD466-3172 VLIGKV3-20*01 87

TABLE 6 Exemplary sequences of TIM-3. SEQ ID NO: Description*Amino acid Sequence  78 Human MFSHLPFDCVLLLLLLLLT TIM-3RSSEVEYRAEVGQNAYLPC immature FYTPAAPGNLVPVCWGKGA proteinCPVFECGNVVLRTDERDVN (Q8TDQ0-1) YWTSRYWLNGDFRKGDVSL TIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAK VTPAPTRQRDFTAAFPRML TTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLAN DLRDSGATIRIGIYIGAGI CAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPP SGLANAVAEGIRSEENIYT IEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP 79 Human SEVEYRAEVGQNAYLPCFY TIM-3 TPAAPGNLVPVCWGKGACPmature VFECGNVVLRTDERDVNYW protein TSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPG IMNDEKFNLKLVIKPAKVT PAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINL TQISTLANELRDSRLANDL RDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHS KEKIQNLSLISLANLPPSG LANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSR QQPSQPLGCRFAMP 101 Human SEVEYRAEVGQNAYLPCFY TIM-3TPAAPGNLVPVCWGKGACP F40A VAECGNVVLRTDERDVNYW TSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPG IMNDEKFNLKLVIKPAKVT PAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINL TQISTLANELRDSRLANDL RDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHS KEKIQNLSLISLANLPPSG LANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSR QQPSQPLGCRFAMP 102 Human SEVEYRAEVGQNAYLPCFY TIM-3TPAAPGNLVPVCWGKGACP fragment VFECGNVVLRTDERDVNYW TSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPG IMNDEKFNLKLVIKPAKVT PAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINL TQISTLANELRDSRLANDL RDSGATIR 93 TIM-3 PVFECGNepitope 94 TIM-3 VCWGKGACPVFECGNVVL epitope 95 TIM-3 RIQIPGIMND epitope96 TIM-3 RIQIPGIMNDEKFNLKL epitope 97 TIM-3 EKFNLKL epitope 98 TIM-3PAAPGNLVP epitope 99 TIM-3 GKGACPVFE epitope 100 TIM-3 DFTAAFPR epitope

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising a VH domain comprising one, two, or all three of theCDRs of a VH domain set forth in Table 1 herein. In certain embodiments,the antibody comprises the CDRH1 of one of VH domains set forth inTable 1. In certain embodiments, the antibody comprises the CDRH2 of oneof the VH domains set forth in Table 1. In certain embodiments, theantibody comprises the CDRH3 of one of the VH domains set forth in Table1.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising a VL domain comprising one, two, or all three of theCDRs of a VL domain disclosed in Table 1 herein. In certain embodiments,the antibody comprises the CDRL1 of one of VL domains set forth inTable 1. In certain embodiments, the antibody comprises the CDRL2 of oneof the VL domains set forth in Table 1. In certain embodiments, theantibody comprises the CDRL3 of one of the VL domains set forth in Table1.

In certain embodiments, the CDRs of an antibody can be determinedaccording to MacCallum R M et al., (1996) J Mol Biol 262: 732-745,herein incorporated by reference in its entirety. See also, e.g., MartinA. “Protein Sequence and Structure Analysis of Antibody VariableDomains,” in Antibody Engineering, Kontermann and Dübel, eds., Chapter31, pp. 422-439, Springer-Verlag, Berlin (2001), herein incorporated byreference in its entirety. In certain embodiments, the heavy chain CDRsof an antibody are determined according to MacCallum and the light chainCDRs of an antibody are determined according to a different method.

In certain embodiments, the CDRs of an antibody can be determinedaccording to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) andKabat et al., Sequences of protein of immunological interest (1991),each of which is herein incorporated by reference in its entirety. Incertain embodiments, the light chain CDRs of an antibody are determinedaccording to Kabat and the heavy chain CDRs of an antibody aredetermined according to MacCallum (supra).

In certain embodiments, the CDRs of an antibody can be determinedaccording to the Chothia numbering scheme, which refers to the locationof immunoglobulin structural loops (see, e.g., Chothia C & Lesk A M,(1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817;Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No.7,709,226, all of which are herein incorporated by reference in theirentireties). Typically, when using the Kabat numbering convention, theChothia CDRH1 loop is present at heavy chain amino acids 26 to 32, 33,or 34, the Chothia CDRH2 loop is present at heavy chain amino acids 52to 56, and the Chothia CDRH3 loop is present at heavy chain amino acids95 to 102, while the Chothia CDRL1 loop is present at light chain aminoacids 24 to 34, the Chothia CDRL2 loop is present at light chain aminoacids 50 to 56, and the Chothia CDRL3 loop is present at light chainamino acids 89 to 97. The end of the Chothia CDRH1 loop when numberedusing the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising the Chothia VH CDRs of a VH disclosed in Table 1herein. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to TIM-3 (e.g., human TIM-3),the antibody comprising the Chothia VL CDRs of a VL disclosed in Table 1herein. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to TIM-3 (e.g., human TIM-3),the antibody comprising the Chothia VH CDRs and Chothia VL CDRs of anantibody disclosed in Table 1 herein. In certain embodiments, antibodiesthat specifically bind to TIM-3 (e.g., human TIM-3) comprise one or moreCDRs, in which the Chothia and Kabat CDRs have the same amino acidsequence. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to TIM-3 (e.g., human TIM-3)and comprises combinations of Kabat CDRs and Chothia CDRs.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the IMGT numbering system as described in Lefranc M-P,(1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999)Nucleic Acids Res 27: 209-212, each of which is herein incorporated byreference in its entirety. According to the IMGT numbering scheme, CDRH1is at positions 26 to 35, CDRH2 is at positions 51 to 57, CDRH3 is atpositions 93 to 102, CDRL1 is at positions 27 to 32, CDRL2 is atpositions 50 to 52, and CDRL3 is at positions 89 to 97.

In certain embodiments, the instant disclosure provides antibodies thatspecifically bind to TIM-3 (e.g., human TIM-3) and comprise CDRs of anantibody disclosed in Table 1 herein, as determined by the IMGTnumbering system, for example, as described in Lefranc M-P (1999) supraand Lefranc M-P et al., (1999) supra.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the AbM numbering scheme, which refers to AbM hypervariableregions, which represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbM antibodymodeling software (Oxford Molecular Group, Inc.), herein incorporated byreference in its entirety. In a particular embodiment, the instantdisclosure provides antibodies that specifically bind to TIM-3 (e.g.,human TIM-3) and comprise CDRs of an antibody disclosed in Table 1herein as determined by the AbM numbering scheme.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinthe antibody comprises a heavy chain variable region comprising theCDRH1, CDRH2, and CDRH3 region amino acid sequences of a VH domain setforth in SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35,and a light chain variable region comprising the CDRL1, CDRL2, and CDRL3region amino acid sequences of a VL domain set forth in SEQ ID NO: 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47, wherein each CDR isdefined in accordance with the MacCallum definition, the Kabatdefinition, the Chothia definition, the combination of the Kabatdefinition and the Chothia definition, the IMGT numbering system, or theAbM definition of CDR.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising:

(a) a CDRH1 comprises the amino acid sequence of X₁X₂X₃X₄X₅S (SEQ ID NO:48), wherein

X₁ is R, S, A, G, K, M, or T,

X₂ is Q, S, A, G, R, or T,

X₃ is N, Y, G, or Q,

X₄ is A or Q, and

X₅ is W, M, A, S, or T; and/or

(b) a CDRH2 comprises the amino acid sequence of WVSAISGSGGSTY (SEQ IDNO: 2); and/or(c) a CDRH3 comprises the amino acid sequence of AKGGDYGGNYFD (SEQ IDNO: 3); and/or(d) a CDRL1 comprises the amino acid sequence of X₁ASQSVX₂SSYLA (SEQ IDNO: 52), wherein

X₁ is R or G, and

X₂ is absent or S; and/or

(e) a CDRL2 comprises the amino acid sequence of X₁ASX₂RAT (SEQ ID NO:53), wherein

X₁ is D or G, and

X₂ is N, S, or T; and/or

(f) a CDRL3 comprises the amino acid sequence of QQYGSSPX₁T (SEQ ID NO:54), wherein X₁ is L or I.

In certain embodiments, CDRH1 comprises the amino acid sequence ofX₁X₂NAWS (SEQ ID NO: 49), wherein: X₁ is R or A; and X₂ is Q or R. Incertain embodiments, CDRH1 comprises the amino acid sequence ofX₁X₂GQX₃S (SEQ ID NO: 50), wherein: X₁ is K, M, or G; X₂ is A or S; andX₃ is S or T. In certain embodiments, CDRH1 comprises the amino acidsequence of X₁X₂QQAS (SEQ ID NO: 51), wherein: X₁ is S, R, T, or G; andX₂ is A, S, T, or G. In certain embodiments, CDRH1 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1, and4-12. In certain embodiments, CDRL1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13-16. In certainembodiments, CDRL2 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 17-21. In certain embodiments, CDRL3comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 22 and 23.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinthe antibody comprises a VH domain comprising the CDRH1, CDRH2 and CDRH3amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3; 4, 2, and 3;5, 2, and 3; 6, 2, and 3; 7, 2, and 3; 8, 2, and 3; 9, 2, and 3; 10, 2,and 3; 11, 2, and 3; or 12, 2, and 3, respectively. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to TIM-3 (e.g., human TIM-3), wherein the antibodycomprises a VH domain comprising the CDRH1, CDRH2 and CDRH3 amino acidsequences set forth in SEQ ID NOs: 1, 2, and 3, respectively. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to TIM-3 (e.g., human TIM-3), wherein the antibodycomprises a VH domain comprising the CDRH1, CDRH2 and CDRH3 amino acidsequences set forth in SEQ ID NOs: 5, 2, and 3, respectively. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to TIM-3 (e.g., human TIM-3), wherein the antibodycomprises a VH domain comprising the CDRH1, CDRH2 and CDRH3 amino acidsequences set forth in SEQ ID NOs: 9, 2, and 3, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinthe antibody comprises a VL domain comprising the CDRL1, CDRL2 and CDRL3amino acid sequences set forth in SEQ ID NOs: 13, 17, and 22; 14, 17,and 22; 15, 18, and 22; 14, 19, and 22; 14, 20, and 22; 14, 21, and 22;16, 20, and 22; or 14, 17, and 23, respectively. In certain embodiments,the instant disclosure provides an isolated antibody that specificallybinds to TIM-3 (e.g., human TIM-3), wherein the antibody comprises a VLdomain comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences setforth in SEQ ID NOs: 14, 21, and 22, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinthe antibody comprises a heavy chain variable region comprising CDRH1,CDRH2, and CDRH3 regions, and a light chain variable region comprisingCDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, and CDRL3 regions comprise the amino acid sequences set forth inSEQ ID NOs: 1, 2, 3, 14, 21, and 22; 4, 2, 3, 14, 21, and 22; 5, 2, 3,14, 21, and 22; 6, 2, 3, 14, 21, and 22; 7, 2, 3, 14, 21, and 22; 8, 2,3, 14, 21, and 22; 9, 2, 3, 14, 21, and 22; 10, 2, 3, 14, 21, and 22;11, 2, 3, 14, 21, and 22; or 12, 2, 3, 14, 21, and 22, respectively. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinthe antibody comprises a heavy chain variable region comprising CDRH1,CDRH2, and CDRH3 regions, and a light chain variable region comprisingCDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, and CDRL3 regions comprise the amino acid sequences set forth inSEQ ID NOs: 1, 2, 3, 14, 21, and 22, respectively. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to TIM-3 (e.g., human TIM-3), wherein the antibodycomprises a heavy chain variable region comprising CDRH1, CDRH2, andCDRH3 regions, and a light chain variable region comprising CDRL1,CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,and CDRL3 regions comprise the amino acid sequences set forth in SEQ IDNOs: 5, 2, 3, 14, 21, and 22, respectively. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto TIM-3 (e.g., human TIM-3), wherein the antibody comprises a heavychain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and alight chain variable region comprising CDRL1, CDRL2, and CDRL3 regions,wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regionscomprise the amino acid sequences set forth in SEQ ID NOs: 9, 2, 3, 14,21, and 22.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 55. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toTIM-3 (e.g., human TIM-3), comprising a heavy chain variable regioncomprising an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99%) identical to the amino acid sequence set forth in SEQ IDNO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In certainembodiments, the antibody comprises a heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, or 35. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 24. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 25. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 26. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 27. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 28. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 29. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 30. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 31. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 32. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 33. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 34. In certain embodiments, the antibodycomprises a heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 35. In certain embodiments, the N-terminalglutamate (E) residue of a heavy chain variable region of an antibody asdescribed herein is replaced with a pyroglutamate (pE) residue.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3),comprising a light chain variable region comprising an amino acidsequence of SEQ ID NO: 56. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toTIM-3 (e.g., human TIM-3), comprising a light chain variable regioncomprising an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99%) identical to the amino acid sequence set forth in SEQ IDNO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In certainembodiments, the antibody comprises a light chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, or 47. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 36. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 37. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 38. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 39. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 40. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 41. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 42. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 43. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 44. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 45. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 46. In certain embodiments, the antibodycomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 47. In certain embodiments, the N-terminalglutamate (E) residue of a light chain variable region of an antibody asdescribed herein is replaced with a pyroglutamate (pE) residue.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 55, and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 56. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto TIM-3 (e.g., human TIM-3), comprising a heavy chain variable regioncomprising an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99%) identical to the amino acid sequence set forth in SEQ IDNO: 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35, and a light chainvariable region comprising an amino acid sequence that is at least 75%,80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence setforth in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47.In certain embodiments, the antibody comprises a heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO: 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, or 35, and a light chain variableregion having the amino acid sequence set forth in SEQ ID NO: 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In certain embodiments, theantibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24 and 43; 26 and43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25 and 42; 25 and41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 24 and 36, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:24 and 38, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 26 and 42, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 24 and 42, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:24 and 46, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 24 and 43, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 26 and 43, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:26 and 46, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 26 and 41, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 24 and 41, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:25 and 39, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 24 and 47, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 25 and 40, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:26 and 47, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 25 and 37, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 25 and 45, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:25 and 44, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 25 and 46, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 25 and 42, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:25 and 41, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 25 and 43, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 25 and 47, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:27 and 46, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 28 and 46, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 29 and 46, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:30 and 46, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 31 and 46, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 32 and 46, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:33 and 46, respectively. In certain embodiments, the antibody comprisesa heavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 34 and 46, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 35 and 46, respectively. In certain embodiments,the N-terminal glutamate (E) residue of a heavy chain variable region ofan antibody as described herein is replaced with a pyroglutamate (pE)residue and/or the N-terminal glutamate (E) residue of a light chainvariable region of the antibody is replaced with a pyroglutamate (pE)residue.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3),comprising a heavy chain variable region having an amino acid sequencederived from a human IGHV3-23 germline sequence (e.g., IGHV3-23*04,e.g., having the amino acid sequence of SEQ ID NO: 84). One or moreregions selected from framework 1, framework 2, framework 3, CDRH1, andCDRH2 (e.g., two, three, four or five of these regions) can be derivedfrom a human IGHV3-23 germline sequence (e.g., IGHV3-23*04, e.g., havingthe amino acid sequence of SEQ ID NO: 84). In one embodiment, framework1, framework 2, framework 3, CDRH1, and CDRH2 are all derived from ahuman IGHV3-23 germline sequence (e.g., IGHV3-23*04, e.g., having theamino acid sequence of SEQ ID NO: 84).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3),comprising a light chain variable region having an amino acid sequencederived from a human germline sequence selected from the groupconsisting of IGKV1-27 (e.g., IGKV1-27*01, e.g., having the amino acidsequence of SEQ ID NO: 85), IGKV3-11 (e.g., IGKV3-11*01, e.g., havingthe amino acid sequence of SEQ ID NO: 86), IGKV3-20 (e.g., IGKV3-20*01,e.g., having the amino acid sequence of SEQ ID NO: 87), and IGKV3D-20(e.g., IGKV3D-20*01, e.g., having the amino acid sequence of SEQ ID NO:88). One or more regions selected from framework 1, framework 2,framework 3, CDRL1, and CDRL2 (e.g., two, three, four or five of theseregions) can be derived from a human germline sequence selected from thegroup consisting of IGKV1-27 (e.g., IGKV1-27*01, e.g., having the aminoacid sequence of SEQ ID NO: 85), IGKV3-11 (e.g., IGKV3-11*01, e.g.,having the amino acid sequence of SEQ ID NO: 86), IGKV3-20 (e.g.,IGKV3-20*01, e.g., having the amino acid sequence of SEQ ID NO: 87), andIGKV3D-20 (e.g., IGKV3D-20*01, e.g., having the amino acid sequence ofSEQ ID NO: 88). In one embodiment, framework 1, framework 2, framework3, CDRL1, and CDRL2 are all derived from a human germline sequenceselected from the group consisting of IGKV1-27 (e.g., IGKV1-27*01, e.g.,having the amino acid sequence of SEQ ID NO: 85), IGKV3-11 (e.g.,IGKV3-11*01, e.g., having the amino acid sequence of SEQ ID NO: 86),IGKV3-20 (e.g., IGKV3-20*01, e.g., having the amino acid sequence of SEQID NO: 87), and IGKV3D-20 (e.g., IGKV3D-20*01, e.g., having the aminoacid sequence of SEQ ID NO: 88).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3),comprising a heavy chain variable region having an amino acid sequencederived from a human IGHV3-23 germline sequence (e.g., IGHV3-23*04,e.g., having the amino acid sequence of SEQ ID NO: 84), and a lightchain variable region having an amino acid sequence derived from a humangermline sequence selected from the group consisting of IGKV1-27 (e.g.,IGKV1-27*01, e.g., having the amino acid sequence of SEQ ID NO: 85),IGKV3-11 (e.g., IGKV3-11*01, e.g., having the amino acid sequence of SEQID NO: 86), IGKV3-20 (e.g., IGKV3-20*01, e.g., having the amino acidsequence of SEQ ID NO: 87), and IGKV3D-20 (e.g., IGKV3D-20*01, e.g.,having the amino acid sequence of SEQ ID NO: 88).

In certain embodiments, the instant disclosure provides an isolatedantibody that cross-competes for binding to TIM-3 (e.g., human TIM-3)with an antibody comprising the heavy and light chain variable regionamino acid sequences set forth in SEQ ID NOs: 24 and 36; 24 and 38; 26and 42; 24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and41; 24 and 41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25and 45; 25 and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and47; 27 and 46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33and 46; 34 and 46; or 35 and 46, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that binds to the same or an overlapping epitope of TIM-3(e.g., an epitope of human TIM-3) as an antibody described herein, e.g.,an antibody comprising the heavy and light chain variable region aminoacid sequences set forth in SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42;24 and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34and 46; or 35 and 46, respectively. In certain embodiments, the epitopeof an antibody can be determined by, e.g., NMR spectroscopy, surfaceplasmon resonance (BIACORE™), X-ray diffraction crystallography studies,ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry(e.g., liquid chromatography electrospray mass spectrometry),array-based oligo-peptide scanning assays, and/or mutagenesis mapping(e.g., site-directed mutagenesis mapping). For X-ray crystallography,crystallization may be accomplished using any of the known methods inthe art (e.g., Giegé R et al., (1994) Acta Crystallogr D BiolCrystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) JBiol Chem 251: 6300-6303, all of which are herein incorporated byreference in their entireties). Antibody:antigen crystals may be studiedusing well known X-ray diffraction techniques and may be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114& 115, eds Wyckoff H W et al.; U.S. Patent Application No.2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D BiolCrystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D BiolCrystallogr 56(Pt 10): 1316-1323, all of which are herein incorporatedby reference in their entireties). Mutagenesis mapping studies may beaccomplished using any method known to one of skill in the art. See,e.g., Champe M et al., (1995) supra and Cunningham B C & Wells J A(1989) supra for a description of mutagenesis techniques, includingalanine scanning mutagenesis techniques. In a specific embodiment, theepitope of an antibody is determined using alanine scanning mutagenesisstudies. In addition, antibodies that recognize and bind to the same oroverlapping epitopes of TIM-3 (e.g., human TIM-3) can be identifiedusing routine techniques such as an immunoassay, for example, by showingthe ability of one antibody to block the binding of another antibody toa target antigen, i.e., a competitive binding assay. Competition bindingassays also can be used to determine whether two antibodies have similarbinding specificity for an epitope. Competitive binding can bedetermined in an assay in which the immunoglobulin under test inhibitsspecific binding of a reference antibody to a common antigen, such asTIM-3 (e.g., human TIM-3). Numerous types of competitive binding assaysare known, for example: solid phase direct or indirect radioimmunoassay(MA), solid phase direct or indirect enzyme immunoassay (EIA), sandwichcompetition assay (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid phase direct biotin-avidin EIA (see Kirkland T N et al.,(1986) J Immunol 137: 3614-9); solid phase direct labeled assay, solidphase direct labeled sandwich assay (see Harlow E & Lane D, (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phasedirect label MA using 1-125 label (see Morel G A et al., (1988) MolImmunol 25(1): 7-15); solid phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176: 546-52); and direct labeled MA (seeMoldenhauer G et al., (1990) Scand J Immunol 32: 77-82), all of whichare herein incorporated by reference in their entireties. Typically,such an assay involves the use of purified antigen (e.g., TIM-3 such ashuman TIM-3) bound to a solid surface or cells bearing either of these,an unlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition can be measured by determining the amount oflabel bound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. A competition bindingassay can be configured in a large number of different formats usingeither labeled antigen or labeled antibody. In a common version of thisassay, the antigen is immobilized on a 96-well plate. The ability ofunlabeled antibodies to block the binding of labeled antibodies to theantigen is then measured using radioactive or enzyme labels. For furtherdetails see, for example, Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68: 269-274;Kuroki M et al., (1990) Cancer Res 50: 4872-4879; Kuroki M et al.,(1992) Immunol Invest 21: 523-538; Kuroki M et al., (1992) Hybridoma 11:391-407 and Antibodies: A Laboratory Manual, Ed Harlow E & Lane Deditors supra, pp. 386-389, all of which are herein incorporated byreference in their entireties.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising a heavy chain comprising the amino acid sequence setforth in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68.In certain embodiments, the antibody comprises a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO: 57. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO: 58. In certain embodiments, theantibody comprises a heavy chain comprising the amino acid sequence setforth in SEQ ID NO: 59. In certain embodiments, the antibody comprises aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:60. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO: 61. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 62. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 63. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 64. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 65. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 66. In certain embodiments,the antibody comprises a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO: 67. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 68. In certain embodiments, the N-terminal glutamate (E)residue of a heavy chain of an antibody as described herein is replacedwith a pyroglutamate (pE) residue.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising a light chain comprising the amino acid sequence setforth in SEQ ID NO: 69. In certain embodiments, the N-terminal glutamate(E) residue of a light chain of an antibody as described herein isreplaced with a pyroglutamate (pE) residue.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 57; and a light chain comprising the amino acid sequence ofSEQ ID NO: 69. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to TIM-3 (e.g., humanTIM-3), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 58; and a light chain comprising the amino acidsequence of SEQ ID NO: 69. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toTIM-3 (e.g., human TIM-3), the antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 59; and a light chaincomprising the amino acid sequence of SEQ ID NO: 69. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to TIM-3 (e.g., human TIM-3), the antibody comprisinga heavy chain comprising the amino acid sequence of SEQ ID NO: 60; and alight chain comprising the amino acid sequence of SEQ ID NO: 69. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 61; and a light chain comprising the amino acid sequence ofSEQ ID NO: 69. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to TIM-3 (e.g., humanTIM-3), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 62; and a light chain comprising the amino acidsequence of SEQ ID NO: 69. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toTIM-3 (e.g., human TIM-3), the antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 63; and a light chaincomprising the amino acid sequence of SEQ ID NO: 69. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to TIM-3 (e.g., human TIM-3), the antibody comprisinga heavy chain comprising the amino acid sequence of SEQ ID NO: 64; and alight chain comprising the amino acid sequence of SEQ ID NO: 69. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 65; and a light chain comprising the amino acid sequence ofSEQ ID NO: 69. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to TIM-3 (e.g., humanTIM-3), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 66; and a light chain comprising the amino acidsequence of SEQ ID NO: 69. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toTIM-3 (e.g., human TIM-3), the antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 67; and a light chaincomprising the amino acid sequence of SEQ ID NO: 69. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to TIM-3 (e.g., human TIM-3), the antibody comprisinga heavy chain comprising the amino acid sequence of SEQ ID NO: 68; and alight chain comprising the amino acid sequence of SEQ ID NO: 69. Incertain embodiments, the N-terminal glutamate (E) residue of a heavychain of an antibody as described herein is replaced with apyroglutamate (pE) residue and/or the N-terminal glutamate (E) residueof a light chain of the antibody is replaced with a pyroglutamate (pE)residue.

Any Ig constant region can be used in the antibodies disclosed herein.In certain embodiments, the Ig region is a human IgG, IgE, IgM, IgD,IgA, or IgY immunoglobulin molecule, any class (e.g., IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂), or any subclass (e.g., IgG_(2a) and IgG_(2b)) ofimmunoglobulin molecule.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising a heavy chain constant region comprising the aminoacid sequence of SEQ ID NO: 70, 71, 72, 73, 74 or 75. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to TIM-3 (e.g., human TIM-3), the antibody comprisinga light chain constant region comprising the amino acid sequence of SEQID NO: 76 or 77.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, numbered according to the EU numbering system, to alter one ormore functional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding and/or antigen-dependentcellular cytotoxicity.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425, herein incorporated by reference in itsentirety. The number of cysteine residues in the hinge region of the CH1domain may be altered to, e.g., facilitate assembly of the light andheavy chains, or to alter (e.g., increase or decrease) the stability ofthe antibody.

In a specific embodiment, one, two, or more amino acid mutations (e.g.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745, all of which are hereinincorporated by reference in their entireties, for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo. In some embodiments, one, two or more amino acidmutations (e.g., substitutions, insertions, or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to decrease the half-lifeof the antibody in vivo. In other embodiments, one, two or more aminoacid mutations (e.g., substitutions, insertions or deletions) areintroduced into an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to increase the half-lifeof the antibody in vivo. In a specific embodiment, the antibodies mayhave one or more amino acid mutations (e.g., substitutions) in thesecond constant (CH2) domain (residues 231-340 of human IgG₁) and/or thethird constant (CH3) domain (residues 341-447 of human IgG₁), numberedaccording to the EU numbering system. In a specific embodiment, theconstant region of the IgG₁ of an antibody described herein comprises amethionine (M) to tyrosine (Y) substitution in position 252, a serine(S) to threonine (T) substitution in position 254, and a threonine (T)to glutamic acid (E) substitution in position 256, numbered according tothe EU numbering system. See U.S. Pat. No. 7,658,921, which is hereinincorporated by reference in its entirety. This type of mutant IgG,referred to as “YTE mutant” has been shown to display fourfold increasedhalf-life as compared to wild-type versions of the same antibody (seeDall'Acqua W F et al., (2006) J Biol Chem 281: 23514-24, which is hereinincorporated by reference in its entirety). In certain embodiments, anantibody comprises an IgG constant domain comprising one, two, three ormore amino acid substitutions of amino acid residues at positions251-257, 285-290, 308-314, 385-389, and 428-436, numbered according tothe EU numbering system.

In some embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, numbered according to the EU numbering system, to increase ordecrease the affinity of the antibody for an Fc receptor (e.g., anactivated Fc receptor) on the surface of an effector cell. Mutations inthe Fc region of an antibody that decrease or increase the affinity ofan antibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, all of which are hereinincorporated by reference in their entireties.

In a further embodiment, one, two, or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322, numbered according to the EU numbering system, can be replaced witha different amino acid residue such that the antibody has an alteredaffinity for an effector ligand but retains the antigen-binding abilityof the parent antibody. The effector ligand to which affinity is alteredcan be, for example, an Fc receptor or the C1 component of complement.This approach is described in further detail in U.S. Pat. Nos. 5,624,821and 5,648,260, each of which is herein incorporated by reference in itsentirety. In some embodiments, the deletion or inactivation (throughpoint mutations or other means) of a constant region domain may reduceFc receptor binding of the circulating antibody thereby increasing tumorlocalization. See, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886, each ofwhich is herein incorporated by reference in its entirety, for adescription of mutations that delete or inactivate the constant domainand thereby increase tumor localization. In certain embodiments, one ormore amino acid substitutions may be introduced into the Fc region of anantibody described herein to remove potential glycosylation sites on Fcregion, which may reduce Fc receptor binding (see, e.g., Shields R L etal., (2001) J Biol Chem 276: 6591-604, which is herein incorporated byreference in its entirety). In various embodiments, one or more of thefollowing mutations in the constant region of an antibody describedherein may be made: an N297A substitution; an N297Q substitution; aL235A substitution and a L237A substitution; a L234A substitution and aL235A substitution; a E233P substitution; a L234V substitution; a L235Asubstitution; a C236 deletion; a P238A substitution; a D265Asubstitution; a A327Q substitution; or a P329A substitution, numberedaccording to the EU numbering system. In certain embodiments, a mutationselected from the group consisting of D265A, P329A, and a combinationthereof, numbered according to the EU numbering system, may be made inthe constant region of an antibody described herein.

In a specific embodiment, an antibody described herein comprises theconstant domain of an IgG₁ with an N297Q or N297A amino acidsubstitution, numbered according to the EU numbering system. In oneembodiment, an antibody described herein comprises the constant domainof an IgG₁ with a mutation selected from the group consisting of D265A,P329A, and a combination thereof, numbered according to the EU numberingsystem. In another embodiment, an antibody described herein comprisesthe constant domain of an IgG₁ with a mutation selected from the groupconsisting of L234A, L235A, and a combination thereof, numberedaccording to the EU numbering system. In certain embodiments, amino acidresidues in the constant region of an antibody described herein in thepositions corresponding to positions L234, L235, and D265 in a humanIgG₁ heavy chain, numbered according to the EU numbering system, are notL, L, and D, respectively. This approach is described in detail inInternational Publication No. WO 14/108483, which is herein incorporatedby reference in its entirety. In a particular embodiment, the aminoacids corresponding to positions L234, L235, and D265 in a human IgG₁heavy chain are F, E, and A; or A, A, and A, respectively, numberedaccording to the EU numbering system.

In certain embodiments, one or more amino acids selected from amino acidresidues 329, 331, and 322 in the constant region of an antibodydescribed herein, numbered according to the EU numbering system, can bereplaced with a different amino acid residue such that the antibody hasaltered C1q binding and/or reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 (Idusogie et al.), which is herein incorporated byreference in its entirety. In some embodiments, one or more amino acidresidues within amino acid positions 231 to 238 in the N-terminal regionof the CH2 domain of an antibody described herein are altered to therebyalter the ability of the antibody to fix complement, numbered accordingto the EU numbering system. This approach is described further inInternational Publication No. WO 94/29351, which is herein incorporatedby reference in its entirety. In certain embodiments, the Fc region ofan antibody described herein is modified to increase the ability of theantibody to mediate antibody dependent cellular cytotoxicity (ADCC)and/or to increase the affinity of the antibody for an Fcγ receptor bymutating one or more amino acids (e.g., introducing amino acidsubstitutions) at the following positions: 238, 239, 248, 249, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309,312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335,337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419,430, 434, 435, 437, 438, or 439, numbered according to the EU numberingsystem. This approach is described further in International PublicationNo. WO 00/42072, which is herein incorporated by reference in itsentirety.

In certain embodiments, an antibody described herein comprises theconstant region of an IgG₄ antibody and the serine at amino acid residue228 of the heavy chain, numbered according to the EU numbering system,is substituted for proline. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toTIM-3 (e.g., human TIM-3), the antibody comprising a heavy chainconstant region comprising the amino acid sequence of SEQ ID NO: 74. Incertain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), theantibody comprising a heavy chain constant region comprising the aminoacid sequence of SEQ ID NO: 75.

In certain embodiments, any of the constant region mutations ormodifications described herein can be introduced into one or both heavychain constant regions of an antibody described herein having two heavychain constant regions.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3) andfunctions as an antagonist.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3) anddecreases TIM-3 (e.g., human TIM-3) activity by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 99% as assessed by methods described herein and/orknown to one of skill in the art, relative to TIM-3 (e.g., human TIM-3)activity without any antibody or with an unrelated antibody (e.g., anantibody that does not specifically bind to TIM-3 (e.g., human TIM-3)).In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3) anddecreases TIM-3 (e.g., human TIM-3) activity by at least about 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold as assessed by methods described herein and/or knownto one of skill in the art, relative to TIM-3 (e.g., human TIM-3)activity without any antibody or with an unrelated antibody (e.g., anantibody that does not specifically bind to TIM-3 (e.g., human TIM-3)).Non-limiting examples of TIM-3 (e.g., human TIM-3) activity can includeTIM-3 (e.g., human TIM-3) signaling, TIM-3 (e.g., human TIM-3) bindingto TIM-3 (e.g., human TIM-3) ligand (e.g., phosphatidylserine), andinhibition of cytokine production (e.g., IFN-γ and/or TNF-α). In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to TIM-3 (e.g., human TIM-3) and deactivates,reduces, or inhibits a TIM-3 (e.g., human TIM-3) activity. In specificembodiments, a decrease in a TIM-3 (e.g., human TIM-3) activity isassessed as described in the Examples, infra.

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3) andreduces TIM-3 (e.g., human TIM-3) binding to its ligand (e.g.,phosphatidylserine) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%,as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, relative to TIM-3 (e.g., human TIM-3)binding to its ligand (e.g., phosphatidylserine) without any antibody orwith an unrelated antibody (e.g., an antibody that does not specificallybind to TIM-3 (e.g., human TIM-3)). In specific embodiments, the instantdisclosure provides an isolated antibody that specifically binds toTIM-3 (e.g., human TIM-3) and reduces TIM-3 (e.g., human TIM-3) bindingto its ligand (e.g., phosphatidylserine) by at least about 1.2 fold, 1.3fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold,4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or100 fold, as assessed by methods described herein (see the Examples,infra) or known to one of skill in the art, relative to TIM-3 (e.g.,human TIM-3) binding to its ligand (e.g., phosphatidylserine) withoutany antibody or with an unrelated antibody (e.g., an antibody that doesnot specifically bind to TIM-3 (e.g., human TIM-3)).

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3) andincreases cytokine production (e.g., IFN-γ and/or TNF-α) by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methodsdescribed herein (see the Examples, infra) or known to one of skill inthe art, relative to cytokine production without any antibody or with anunrelated antibody (e.g., an antibody that does not specifically bind toTIM-3 (e.g., human TIM-3)). In specific embodiments, the instantdisclosure provides an isolated antibody that specifically binds toTIM-3 (e.g., human TIM-3) and increases cytokine production (e.g., IFN-γand/or TNF-α) by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold,2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, as assessed bymethods described herein (see the Examples, infra) or known to one ofskill in the art, relative to cytokine production without any antibodyor with an unrelated antibody (e.g., an antibody that does notspecifically bind to TIM-3 (e.g., human TIM-3)).

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3) and eitheralone or in combination with an anti-PD-1 antibody (e.g., pembrolizumabor nivolumab) increases IFN-γ production in human peripheral bloodmononuclear cells (PBMCs) in response to Staphylococcus Enterotoxin A(SEA) stimulation by at least about 1.2 fold, 1.3 fold, 1.4 fold, 1.5fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, asassessed by methods described herein (see the Examples, infra) or knownto one of skill in the art, relative to IFN-γ production without anyantibody or with an unrelated antibody (e.g., an antibody that does notspecifically bind to TIM-3 (e.g., human TIM-3)).

In certain embodiments, human peripheral blood mononuclear cells (PBMCs)stimulated with Staphylococcus Enterotoxin A (SEA) in the presence of anantibody described herein, which specifically binds to TIM-3 (e.g.,human TIM-3), have increased IFN-γ production by at least about 1.2fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold,4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold relative to PBMCs only stimulated with SEA without anyantibody or with an unrelated antibody (e.g., an antibody that does notspecifically bind to TIM-3 (e.g., human TIM-3)), as assessed by methodsdescribed herein (see the Examples, infra) or known to one of skill inthe art.

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3) and eitheralone or in combination with an anti-PD-1 antibody (e.g., pembrolizumabor nivolumab) increases IFN-γ and/or TNFα production in tumorinfiltrating lymphocytes (TILs) in response to anti-CD3 antibody andanti-CD28 antibody stimulation by at least about 1.2 fold, 1.3 fold, 1.4fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold,as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, relative to IFN-γ and/or TNFαproduction without an antibody that specifically binds to TIM-3 (e.g.,human TIM-3). In one embodiment, the TILs are from non-small cell lungcancer (NSCLC) tumor. In another embodiment, the TILs are fromgallbladder adenocarcinoma tumor. In another embodiment, the TILs arefrom breast cancer tumor.

In certain embodiments, tumor infiltrating lymphocytes (TILs) stimulatedwith anti-CD3 and anti-CD28 antibodies in the presence of an antibodydescribed herein, which specifically binds to TIM-3 (e.g., human TIM-3),have increased IFN-γ and/or TNFα production by at least about 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold relative to TILs only stimulated with anti-CD3 andanti-CD28 antibodies without an antibody that specifically binds toTIM-3 (e.g., human TIM-3), as assessed by methods described herein (seethe Examples, infra) or known to one of skill in the art. In oneembodiment, the TILs are from non-small cell lung cancer (NSCLC) tumor.In another embodiment, the TILs are from gallbladder adenocarcinomatumor. In another embodiment, the TILs are from breast cancer tumor.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3) and isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3).In specific embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or99% of the antibody described herein is internalized upon binding tocells expressing TIM-3 (e.g., human TIM-3), as assessed by methodsdescribed herein (see the Examples, infra) or known to one of skill inthe art. In certain embodiments, a lower percentage of the cellsexpressing TIM-3 (e.g., human TIM-3) survive in the presence of theantibody described herein than in the presence of a reference anti-TIM-3(e.g., human TIM-3) antibody in an assay comprising the following steps:

(a) plating the cells expressing TIM-3 (e.g., human TIM-3) at 2×10⁴cells per well in a tissue culture plate;(b) adding the same concentrations of αHFc-NC-DM1 and the antibodydescribed herein or the reference anti-TIM-3 (e.g., human TIM-3)antibody (e.g., 1.5 ng/ml, 4.6 ng/ml, 13.7 ng/ml, 41.2 ng/ml, 123.5ng/ml, 370 ng/ml, 1111 ng/ml, or 3333 ng/ml) at a final volume of 100μl/well;(c) incubating at 37° C. and 5% CO₂ for 72 hours;(d) measuring survival of the cells expressing TIM-3 (e.g., humanTIM-3); and(e) calculating percentage of cell survival relative to untreated cellsexpressing TIM-3 (e.g., human TIM-3).In certain embodiments, the percentage of cell survival in the presenceof the antibody described herein is at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 99% lower than the percentage of cell survival in thepresence of the reference anti-TIM-3 (e.g., human TIM-3) antibody Incertain embodiments, the percentage of cell survival in the presence ofthe antibody described herein is at least about 1.2 fold, 1.3 fold, 1.4fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 foldlower than the percentage of cell survival in the presence of thereference anti-TIM-3 (e.g., human TIM-3) antibody. In certainembodiments, the reference anti-TIM-3 (e.g., human TIM-3) antibody ispab1944w (IgG₁ N297A). In certain embodiments, the reference anti-TIM-3(e.g., human TIM-3) antibody is Hum11 (IgG₄ S228P). In certainembodiments, the cells expressing TIM-3 (e.g., human TIM-3) are Kasumi-3cells. In certain embodiments, the cells expressing TIM-3 (e.g., humanTIM-3) are Kasumi-3 cells (ATCC® CRL-2725™). In certain embodiments, thecells expressing TIM-3 (e.g., human TIM-3) are Jurkat cells engineeredto express TIM-3 (e.g., human TIM-3).

In certain embodiments, at most 50% of the cells expressing TIM-3 (e.g.,human TIM-3) survive in the presence of the antibody described hereinrelative to untreated cells expressing TIM-3 (e.g., human TIM-3) in anassay comprising the following steps:

(a) plating the cells expressing TIM-3 (e.g., human TIM-3) at 2×10⁴cells per well in a tissue culture plate;(b) adding the same concentrations of αHFc-NC-DM1 and the antibodydescribed herein (e.g., 1.5 ng/ml, 4.6 ng/ml, 13.7 ng/ml, 41.2 ng/ml,123.5 ng/ml, 370 ng/ml, 1111 ng/ml, or 3333 ng/ml) at a final volume of100 μl/well;(c) incubating at 37° C. and 5% CO₂ for 72 hours;(d) measuring survival of the cells expressing TIM-3 (e.g., humanTIM-3); and(e) calculating percentage of cell survival relative to untreated cellsexpressing TIM-3 (e.g., human TIM-3).In certain embodiments, at most 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% of the cells expressing TIM-3 (e.g., human TIM-3) survive in thepresence of the antibody described herein relative to untreated cellsexpressing TIM-3 (e.g., human TIM-3). In certain embodiments,αHFc-NC-DM1 and the antibody described herein are added at aconcentration of 1111 ng/ml and at most 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, or 50% of the cells expressing TIM-3 (e.g., human TIM-3)survive in the presence of the antibody described herein relative tountreated cells expressing TIM-3 (e.g., human TIM-3). In certainembodiments, αHFc-NC-DM1 and the antibody described herein are added ata concentration of 1111 ng/ml and at most 50% of the cells expressingTIM-3 (e.g., human TIM-3) survive in the presence of the antibodydescribed herein relative to untreated cells expressing TIM-3 (e.g.,human TIM-3).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody comprises aCDRH1 comprising the amino acid sequence of X₁X₂X₃X₄X₅S (SEQ ID NO: 48),wherein

X₁ is R, S, A, G, K, M, or T,

X₂ is Q, S, A, G, R, or T,

X₃ is N, Y, G, or Q,

X₄ is A or Q, and

X₅ is W, M, A, S, or T.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody comprises aCDRH1 comprising the amino acid sequence of X₁X₂NAWS (SEQ ID NO: 49),wherein

X₁ is R or A; and

X₂ is Q or R.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody comprises aCDRH1 comprising the amino acid sequence of X₁X₂GQX₃S (SEQ ID NO: 50),wherein

X₁ is K, M, or G;

X₂ is A or S; and

X₃ is S or T.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody comprises aCDRH1 comprising the amino acid sequence of X₁X₂QQAS (SEQ ID NO: 51),wherein

X₁ is S, R, T, or G; and

X₂ is A, S, T, or G.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody comprises aCDRH2 comprising the amino acid sequence of WVSAISGSGGSTY (SEQ ID NO:2).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody comprises aCDRH3 comprising the amino acid sequence of AKGGDYGGNYFD (SEQ ID NO: 3).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody comprises aCDRL1 comprising the amino acid sequence of X₁ASQSVX₂SSYLA (SEQ ID NO:52), wherein

X₁ is R or G, and

X₂ is absent or S.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody comprises aCDRL2 comprising the amino acid sequence of X₁ASX₂RAT (SEQ ID NO: 53),wherein

X₁ is D or G, and

X₂ is N, S, or T.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody comprises aCDRL3 comprising the amino acid sequence of QQYGSSPX₁T (SEQ ID NO: 54),wherein

X₁ is L or I.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibodycross-competes for binding to TIM-3 (e.g., human TIM-3) with an antibodycomprising the heavy and light chain variable region amino acidsequences set forth in SEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24and 42; 24 and 46; 24 and 43; 26 and 43; 26 and 46; 26 and 41; 24 and41; 25 and 39; 24 and 47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25and 44; 25 and 46; 25 and 42; 25 and 41; 25 and 43; 25 and 47; 27 and46; 28 and 46; 29 and 46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34and 46; or 35 and 46, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody binds to thesame or an overlapping epitope of TIM-3 (e.g., an epitope of humanTIM-3) as an antibody described herein, e.g., an antibody comprising theheavy and light chain variable region amino acid sequences set forth inSEQ ID NOs: 24 and 36; 24 and 38; 26 and 42; 24 and 42; 24 and 46; 24and 43; 26 and 43; 26 and 46; 26 and 41; 24 and 41; 25 and 39; 24 and47; 25 and 40; 26 and 47; 25 and 37; 25 and 45; 25 and 44; 25 and 46; 25and 42; 25 and 41; 25 and 43; 25 and 47; 27 and 46; 28 and 46; 29 and46; 30 and 46; 31 and 46; 32 and 46; 33 and 46; 34 and 46; or 35 and 46,respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to TIM-3 (e.g., human TIM-3), whereinat least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the antibody isinternalized upon binding to cells expressing TIM-3 (e.g., human TIM-3),as assessed by methods described herein (see the Examples, infra) orknown to one of skill in the art, and wherein the antibody comprises ahuman IgG heavy chain constant region that is a variant of a wild typehuman IgG heavy chain constant region, wherein the variant human IgGheavy chain constant region binds to a human Fc gamma receptor withlower affinity than the wild type human IgG heavy chain constant regionbinds to the human Fc gamma receptor. In certain embodiments, the humanFc gamma receptor is selected from the group consisting of FcγRI,FcγRII, and FcγRIII. In certain embodiments, the variant human IgG heavychain constant region is an IgG₁ constant region comprising a N297Amutation, numbered according to the EU numbering system.

5.3 Pharmaceutical Compositions

Provided herein are compositions comprising an anti-TIM-3 (e.g., humanTIM-3) antibody described herein having the desired degree of purity ina physiologically acceptable carrier, excipient or stabilizer(Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton,Pa.). Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In a specific embodiment, pharmaceutical compositions comprise ananti-TIM-3 (e.g., human TIM-3) antibody described herein, and optionallyone or more additional prophylactic or therapeutic agents, in apharmaceutically acceptable carrier. In a specific embodiment,pharmaceutical compositions comprise an effective amount of an antibodydescribed herein, and optionally one or more additional prophylactic ortherapeutic agents, in a pharmaceutically acceptable carrier. In someembodiments, the antibody is the only active ingredient included in thepharmaceutical composition. Pharmaceutical compositions described hereincan be useful in inhibiting TIM-3 (e.g., human TIM-3) activity andtreating a condition, such as cancer or an infectious disease. In oneembodiment, the present invention relates to a pharmaceuticalcomposition of the present invention comprising an anti-TIM-3 antibodyof the present invention for use as a medicament. In another embodiment,the present invention relates to a pharmaceutical composition of thepresent invention for use in a method for the treatment of cancer or aninfectious disease. In another embodiment, the present invention relatesto use of a pharmaceutical composition of the invention for preparing amedicament for treating cancer or an infectious disease.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

A pharmaceutical composition may be formulated for any route ofadministration to a subject. Specific examples of routes ofadministration include intranasal, oral, pulmonary, transdermal,intradermal, and parenteral. Parenteral administration, characterized byeither subcutaneous, intramuscular or intravenous injection, is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of an antibody includesterile solutions ready for injection, sterile dry soluble products,such as lyophilized powders, ready to be combined with a solvent justprior to use, including hypodermic tablets, sterile suspensions readyfor injection, sterile dry insoluble products ready to be combined witha vehicle just prior to use and sterile emulsions. The solutions may beeither aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Topical mixtures comprising an antibody are prepared as described forthe local and systemic administration. The resulting mixture can be asolution, suspension, emulsions or the like and can be formulated ascreams, gels, ointments, emulsions, solutions, elixirs, lotions,suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,suppositories, bandages, dermal patches or any other formulationssuitable for topical administration.

An anti-TIM-3 (e.g., human TIM-3) antibody described herein can beformulated as an aerosol for topical application, such as by inhalation(see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209 and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatment ofinflammatory diseases, particularly asthma and are herein incorporatedby reference in their entireties). These formulations for administrationto the respiratory tract can be in the form of an aerosol or solutionfor a nebulizer, or as a microtine powder for insufflations, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will, in one embodiment, have diameters ofless than 50 microns, in one embodiment less than 10 microns.

An anti-TIM-3 (e.g., human TIM-3) antibody described herein can beformulated for local or topical application, such as for topicalapplication to the skin and mucous membranes, such as in the eye, in theform of gels, creams, and lotions and for application to the eye or forintracisternal or intraspinal application. Topical administration iscontemplated for transdermal delivery and also for administration to theeyes or mucosa, or for inhalation therapies. Nasal solutions of theantibody alone or in combination with other pharmaceutically acceptableexcipients can also be administered.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art, and can be used toadminister an antibody. For example, such patches are disclosed in U.S.Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975,6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of whichare herein incorporated by reference in their entireties.

In certain embodiments, a pharmaceutical composition comprising anantibody described herein is a lyophilized powder, which can bereconstituted for administration as solutions, emulsions and othermixtures. It may also be reconstituted and formulated as solids or gels.The lyophilized powder is prepared by dissolving an antibody describedherein, or a pharmaceutically acceptable derivative thereof, in asuitable solvent. In some embodiments, the lyophilized powder issterile. The solvent may contain an excipient which improves thestability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbitol, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

The anti-TIM-3 (e.g., human TIM-3) antibodies described herein and othercompositions provided herein can also be formulated to be targeted to aparticular tissue, receptor, or other area of the body of the subject tobe treated. Many such targeting methods are well known to those of skillin the art. All such targeting methods are contemplated herein for usein the instant compositions. For non-limiting examples of targetingmethods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,5,840,674, 5,759,542 and 5,709,874, all of which are herein incorporatedby reference in their entireties. In a specific embodiment, an antibodydescribed herein is targeted to a tumor.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

5.4 Methods of Use and Uses

In another aspect, the instant disclosure provides a method of treatinga subject using the anti-TIM-3 (e.g., human TIM-3) antibodies disclosedherein. Any disease or disorder in a subject that would benefit frominhibition of TIM-3 (e.g., human TIM-3) function can be treated usingthe anti-TIM-3 (e.g., human TIM-3) antibodies disclosed herein. Theanti-TIM-3 (e.g., human TIM-3) antibodies disclosed herein areparticularly useful for inhibiting immune system tolerance to tumors,and accordingly can be used as an immunotherapy for subjects withcancer. For example, in certain embodiments, the instant disclosureprovides a method of increasing T cell activation in response to anantigen in a subject, the method comprising administering to the subjectan effective amount of an anti-TIM-3 (e.g., human TIM-3) antibody orpharmaceutical composition thereof, as disclosed herein. In certainembodiments, the instant disclosure provides a method of treating cancerin a subject, the method comprising administering to the subject aneffective amount of the antibody or pharmaceutical composition, asdisclosed herein. In certain embodiments, the instant disclosureprovides an antibody or pharmaceutical composition as disclosed hereinfor use in a method for the treatment of cancer or an infectiousdisease. In certain embodiments, the instant disclosure provides anantibody or pharmaceutical composition as disclosed herein for use as amedicament. In another embodiment, the instant disclosure provides useof an antibody or pharmaceutical composition as disclosed herein forpreparing a medicament for treating cancer or an infectious disease.

Cancers that can be treated with the anti-TIM-3 (e.g., human TIM-3)antibodies or pharmaceutical compositions disclosed herein include,without limitation, a solid tumor, a hematological cancer (e.g.,leukemia, lymphoma, myeloma, e.g., multiple myeloma), and a metastaticlesion. In one embodiment, the cancer is a solid tumor. Examples ofsolid tumors include malignancies, e.g., sarcomas and carcinomas, e.g.,adenocarcinomas of the various organ systems, such as those affectingthe lung, breast, ovarian, lymphoid, gastrointestinal (e.g., colon),anal, genitals and genitourinary tract (e.g., renal, urothelial, bladdercells, prostate), pharynx, CNS (e.g., brain, neural or glial cells),head and neck, skin (e.g., melanoma), and pancreas, as well asadenocarcinomas which include malignancies such as colon cancers, rectalcancer, renal-cell carcinoma, liver cancer, lung cancer (e.g., non-smallcell lung cancer or small cell lung cancer), cancer of the smallintestine and cancer of the esophagus. The cancer may be at an early,intermediate, late stage or metastatic cancer. In certain embodiments,the cancer is associated with elevated PD-1 activity (e.g., elevatedPD-1 expression).

In one embodiment, the cancer is chosen from a lung cancer (e.g., lungadenocarcinoma or a non-small cell lung cancer (NSCLC) (e.g., a NSCLCwith squamous and/or non-squamous histology, or a NSCLCadenocarcinoma)), a melanoma (e.g., an advanced melanoma), a renalcancer (e.g., a renal cell carcinoma), a liver cancer (e.g.,hepatocellular carcinoma), a myeloma (e.g., a multiple myeloma), aprostate cancer, a breast cancer (e.g., a breast cancer that does notexpress one, two or all of estrogen receptor, progesterone receptor, orHer2/neu, e.g., a triple negative breast cancer), an ovarian cancer, acolorectal cancer, a pancreatic cancer, a head and neck cancer (e.g.,head and neck squamous cell carcinoma (HNSCC), anal cancer,gastro-esophageal cancer (e.g., esophageal squamous cell carcinoma),mesothelioma, nasopharyngeal cancer, thyroid cancer, cervical cancer,epithelial cancer, peritoneal cancer, or a lymphoproliferative disease(e.g., a post-transplant lymphoproliferative disease). In oneembodiment, the cancer is NSCLC. In one embodiment, the cancer is arenal cell carcinoma. In one embodiment, the cancer is an ovariancancer. In a specific embodiment, the ovarian cancer is aplatinum-refractory ovarian cancer.

In one embodiment, the cancer is a hematological cancer, for example, aleukemia, a lymphoma, or a myeloma. In one embodiment, the cancer is aleukemia, for example, acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), acute myeloblastic leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CIVIL),chronic myeloid leukemia (CIVIL), chronic myelomonocytic leukemia(CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. Inone embodiment, the cancer is a lymphoma, for example, B cell lymphoma,diffuse large B-cell lymphoma (DLBCL), activated B-cell like (ABC)diffuse large B cell lymphoma, germinal center B cell (GCB) diffuselarge B cell lymphoma, mantle cell lymphoma, Hodgkin lymphoma,non-Hodgkin lymphoma, relapsed non-Hodgkin lymphoma, refractorynon-Hodgkin lymphoma, recurrent follicular non-Hodgkin lymphoma, Burkittlymphoma, small lymphocytic lymphoma, follicular lymphoma,lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma. In oneembodiment the cancer is a myeloma, for example, multiple myeloma.

In another embodiment, the cancer is chosen form a carcinoma (e.g.,advanced or metastatic carcinoma), melanoma or a lung carcinoma, e.g., anon-small cell lung carcinoma.

In one embodiment, the cancer is a lung cancer, e.g., a lungadenocarcinoma, non-small cell lung cancer or small cell lung cancer.

In one embodiment, the cancer is a melanoma, e.g., an advanced melanoma.In one embodiment, the cancer is an advanced or unresectable melanomathat does not respond to other therapies. In other embodiments, thecancer is a melanoma with a BRAF mutation (e.g., a BRAF V600 mutation).In yet other embodiments, the anti-TIM-3 (e.g., human TIM-3) antibody orpharmaceutical composition disclosed herein is administered aftertreatment with an anti-CTLA-4 antibody (e.g., ipilimumab) with orwithout a BRAF inhibitor (e.g., vemurafenib or dabrafenib).

In another embodiment, the cancer is a hepatocarcinoma, e.g., anadvanced hepatocarcinoma, with or without a viral infection, e.g., achronic viral hepatitis.

In another embodiment, the cancer is a prostate cancer, e.g., anadvanced prostate cancer.

In yet another embodiment, the cancer is a myeloma, e.g., multiplemyeloma.

In yet another embodiment, the cancer is a renal cancer, e.g., a renalcell carcinoma (RCC) (e.g., a metastatic RCC, clear cell renal cellcarcinoma (CCRCC) or kidney papillary cell carcinoma).

In yet another embodiment, the cancer is chosen from a lung cancer, amelanoma, a renal cancer, a breast cancer, a colorectal cancer, aleukemia, or a metastatic lesion of the cancer.

In certain embodiments, the instant disclosure provides a method ofpreventing or treating an infectious disease in a subject, the methodcomprising administering to the subject an effective amount of ananti-TIM-3 (e.g., human TIM-3) antibody or pharmaceutical compositionthereof, as disclosed herein. In one embodiment, provided herein aremethods for preventing and/or treating an infection (e.g., a viralinfection, a bacterial infection, a fungal infection, a protozoalinfection, or a parasitic infection). The infection prevented and/ortreated in accordance with the methods can be caused by an infectiousagent identified herein. In a specific embodiment, an anti-TIM-3 (e.g.,human TIM-3) antibody described herein or a composition thereof is theonly active agent administered to a subject. In some embodiments, ananti-TIM-3 (e.g., human TIM-3) antibody described herein or acomposition thereof is used in combination with anti-infectiveinterventions (e.g., antivirals, antibacterials, antifungals, oranti-helminthics) for the treatment of infectious diseases. Therefore,in a one embodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention for use in a methodof preventing and/or treating an infectious disease, optionally whereinthe antibody or pharmaceutical composition is the only active agentadministered to a subject, or wherein the antibody or pharmaceuticalcomposition is used in combination with anti-infective interventions.

Infectious diseases that can be treated and/or prevented by anti-TIM-3(e.g., human TIM-3) antibodies or pharmaceutical compositions disclosedherein are caused by infectious agents including but not limited tobacteria, parasites, fungi, protozae, and viruses. In a specificembodiment, the infectious disease treated and/or prevented byanti-TIM-3 (e.g., human TIM-3) antibodies or pharmaceutical compositionsdisclosed herein is caused by a virus. Viral diseases or viralinfections that can be prevented and/or treated in accordance with themethods described herein include, but are not limited to, those causedby hepatitis type A, hepatitis type B, hepatitis type C, influenza(e.g., influenza A or influenza B), varicella, adenovirus, herpessimplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest,rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papillomavirus, papova virus, cytomegalovirus, echinovirus, arbovirus,huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus,polio virus, small pox, Epstein Barr virus, human immunodeficiency virustype I (HIV-I), human immunodeficiency virus type II (HIV-II), andagents of viral diseases such as viral meningitis, encephalitis, dengueor small pox.

Bacterial infections that can be prevented and/or treated includeinfections caused by Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa. Bacterial diseasescaused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa) that can beprevented and/or treated in accordance with the methods described hereininclude, but are not limited to, Mycobacteria rickettsia, Mycoplasma,Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillusantracis (anthrax), tetanus, Streptococcus, Staphylococcus,mycobacterium, pertissus, cholera, plague, diptheria, chlamydia, S.aureus and legionella.

Protozoal diseases or protozoal infections caused by protozoa that canbe prevented and/or treated in accordance with the methods describedherein include, but are not limited to, leishmania, coccidiosis,trypanosoma schistosoma or malaria. Parasitic diseases or parasiticinfections caused by parasites that can be prevented and/or treated inaccordance with the methods described herein include, but are notlimited to, chlamydia and rickettsia.

Fungal diseases or fungal infections that can be prevented and/ortreated in accordance with the methods described herein include, but arenot limited to, those caused by Candida infections, zygomycosis, Candidamastitis, progressive disseminated trichosporonosis with latenttrichosporonemia, disseminated candidiasis, pulmonaryparacoccidioidomycosis, pulmonary aspergillosis, Pneumocystis cariniipneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis andcerebrospinal vasculitis, Aspergillus niger infection, Fusariumkeratitis, paranasal sinus mycoses, Aspergillus fumigatus endocarditis,tibial dyschondroplasia, Candida glabrata vaginitis, oropharyngealcandidiasis, X-linked chronic granulomatous disease, tinea pedis,cutaneous candidiasis, mycotic placentitis, disseminatedtrichosporonosis, allergic bronchopulmonary aspergillosis, mycotickeratitis, Cryptococcus neoformans infection, fungal peritonitis,Curvularia geniculata infection, staphylococcal endophthalmitis,sporotrichosis, and dermatophytosis.

In certain embodiments, these methods further comprise administering anadditional therapeutic agent to the subject. In certain embodiments, theadditional therapeutic agent is a chemotherapeutic, a radiotherapeutic,or a checkpoint targeting agent. In certain embodiments, thechemotherapeutic agent is a hypomethylating agent (e.g., azacitidine).In certain embodiments, the checkpoint targeting agent is selected fromthe group consisting of an antagonist anti-CTLA-4 antibody, anantagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, anantagonist anti-PD-1 antibody, an antagonist anti-TIM-3 antibody, anantagonist anti-LAG-3 antibody, an antagonist anti-CEACAM1 antibody, anagonist anti-CD137 antibody, an antagonist anti-TIGIT antibody, anantagonist anti-VISTA antibody, an agonist anti-GITR antibody, and anagonist anti-0X₄₀ antibody.

In one embodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention for use in a methodof the present invention, wherein the method further comprisesadministering an additional therapeutic agent to the subject. In oneembodiment, the present invention relates to (a) an antibody and/orpharmaceutical composition of the present invention and (b) anadditional therapeutic agent for use as a medicament. In one embodiment,the present invention relates to (a) an antibody and/or pharmaceuticalcomposition of the present invention, and (b) an additional therapeuticagent for use in a method for the treatment of cancer. In a furtherembodiment, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody and/orpharmaceutical composition of the present invention and (b) anadditional therapeutic agent. In one embodiment, the additionaltherapeutic agent is a chemotherapeutic, a radiotherapeutic, or acheckpoint targeting agent.

In certain embodiments, an anti-PD-1 antibody is used in methodsdisclosed herein. In certain embodiments, the anti-PD-1 antibody isnivolumab, also known as BMS-936558 or MDX1106, developed byBristol-Myers Squibb. In certain embodiments, the anti-PD-1 antibody ispembrolizumab, also known as lambrolizumab or MK-3475, developed byMerck & Co. In certain embodiments, the anti-PD-1 antibody ispidilizumab, also known as CT-011, developed by CureTech. In certainembodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514,developed by Medimmune. In certain embodiments, the anti-PD-1 antibodyis PDR001 developed by Novartis Pharmaceuticals. In certain embodiments,the anti-PD-1 antibody is REGN2810 developed by RegeneronPharmaceuticals. In certain embodiments, the anti-PD-1 antibody isPF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1antibody is BGB-A317 developed by BeiGene. In certain embodiments, theanti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. Incertain embodiments, the anti-PD-1 antibody is SHR-1210 developed byHengrui.

Further non-limiting examples of anti-PD-1 antibodies that may be usedin treatment methods disclosed herein are disclosed in the followingpatents and patent applications, all of which are herein incorporated byreference in their entireties for all purposes: U.S. Pat. Nos.6,808,710; 7,332,582; 7,488,802; 8,008,449; 8,114,845; 8,168,757;8,354,509; 8,686,119; 8,735,553; 8,747,847; 8,779,105; 8,927,697;8,993,731; 9,102,727; 9,205,148; U.S. Publication No. US 2013/0202623A1; U.S. Publication No. US 2013/0291136 A1; U.S. Publication No. US2014/0044738 A1; U.S. Publication No. US 2014/0356363 A1; U.S.Publication No. US 2016/0075783 A1; and PCT Publication No. WO2013/033091 A1; PCT Publication No. WO 2015/036394 A1; PCT PublicationNo. WO 2014/179664 A2; PCT Publication No. WO 2014/209804 A1; PCTPublication No. WO 2014/206107 A1; PCT Publication No. WO 2015/058573A1; PCT Publication No. WO 2015/085847 A1; PCT Publication No. WO2015/200119 A1; PCT Publication No. WO 2016/015685 A1; and PCTPublication No. WO 2016/020856 A1.

In certain embodiments, an anti-PD-L1 antibody is used in methodsdisclosed herein. In certain embodiments, the anti-PD-L1 antibody isatezolizumab developed by Genentech. In certain embodiments, theanti-PD-L1 antibody is durvalumab developed by AstraZeneca, Celgene andMedimmune. In certain embodiments, the anti-PD-L1 antibody is avelumab,also known as MSB0010718C, developed by Merck Serono and Pfizer. Incertain embodiments, the anti-PD-L1 antibody is MDX-1105 developed byBristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody isAMP-224 developed by Amplimmune and GSK.

Non-limiting examples of anti-PD-L1 antibodies that may be used intreatment methods disclosed herein are disclosed in the followingpatents and patent applications, all of which are herein incorporated byreference in their entireties for all purposes: U.S. Pat. Nos.7,943,743; 8,168,179; 8,217,149; 8,552,154; 8,779,108; 8,981,063;9,175,082; U.S. Publication No. US 2010/0203056 A1; U.S. Publication No.US 2003/0232323 A1; U.S. Publication No. US 2013/0323249 A1; U.S.Publication No. US 2014/0341917 A1; U.S. Publication No. US 2014/0044738A1; U.S. Publication No. US 2015/0203580 A1; U.S. Publication No. US2015/0225483 A1; U.S. Publication No. US 2015/0346208 A1; U.S.Publication No. US 2015/0355184 A1; and PCT Publication No. WO2014/100079 A1; PCT Publication No. WO 2014/022758 A1; PCT PublicationNo. WO 2014/055897 A2; PCT Publication No. WO 2015/061668 A1; PCTPublication No. WO 2015/109124 A1; PCT Publication No. WO 2015/195163A1; PCT Publication No. WO 2016/000619 A1; and PCT Publication No. WO2016/030350 A1.

In certain embodiments, an anti-TIM-3 (e.g., human TIM-3) antibodydisclosed herein is administered to a subject in combination with acompound that targets an immunomodulatory enzyme(s) such as IDO(indoleamine-(2,3)-dioxygenase) and/or TDO (tryptophan 2,3-dioxygenase).Therefore, in one embodiment, the additional therapeutic agent is acompound that targets an immunomodulatory enzyme(s), such as aninhibitor of indoleamine-(2,3)-dioxygenase (IDO). In certainembodiments, such compound is selected from the group consisting ofepacadostat (Incyte Corp; see, e.g., WO 2010/005958 which is hereinincorporated by reference in its entirety), F001287 (FlexusBiosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), andNLG919 (NewLink Genetics). In one embodiment, the compound isepacadostat. In another embodiment, the compound is F001287. In anotherembodiment, the compound is indoximod. In another embodiment, thecompound is NLG919. In a specific embodiment, an anti-TIM-3 (e.g., humanTIM-3) antibody disclosed herein is administered to a subject incombination with an IDO inhibitor for treating cancer. The IDO inhibitoras described herein for use in treating cancer is present in a soliddosage form of a pharmaceutical composition such as a tablet, a pill ora capsule, wherein the pharmaceutical composition includes an IDOinhibitor and a pharmaceutically acceptable excipient. As such, theantibody as described herein and the IDO inhibitor as described hereincan be administered separately, sequentially or concurrently as separatedosage forms. In one embodiment, the antibody is administeredparenterally, and the IDO inhibitor is administered orally. Inparticular embodiments, the inhibitor is selected from the groupconsisting of epacadostat (Incyte Corporation), F001287 (FlexusBiosciences/Bristol-Myers Squibb), indoximod (NewLink Genetics), andNLG919 (NewLink Genetics). Epacadostat has been described in PCTPublication No. WO 2010/005958, which is herein incorporated byreference in its entirety for all purposes. In one embodiment, theinhibitor is epacadostat. In another embodiment, the inhibitor isF001287. In another embodiment, the inhibitor is indoximod. In anotherembodiment, the inhibitor is NLG919.

In certain embodiments, an anti-TIM-3 (e.g., human TIM-3) antibodydisclosed herein is administered to a subject in combination with avaccine. The vaccine can be, e.g., a peptide vaccine, a DNA vaccine, oran RNA vaccine. In certain embodiments, the vaccine is a heat shockprotein based tumor vaccine or a heat shock protein based pathogenvaccine. In certain embodiments, an anti-TIM-3 (e.g., human TIM-3)antibody disclosed herein is administered to a subject in combinationwith a vaccine as described in WO 2016/183486 (e.g., a vaccinecomprising at least one synthetic peptide comprising a cancer-specificmutation present in a cancer from the subject), incorporated herein byreference in its entirety. In a specific embodiment, an anti-TIM-3(e.g., human TIM-3) antibody disclosed herein is administered to asubject in combination with a heat shock protein based tumor-vaccine.Heat shock proteins (HSPs) are a family of highly conserved proteinsfound ubiquitously across all species. Their expression can bepowerfully induced to much higher levels as a result of heat shock orother forms of stress, including exposure to toxins, oxidative stress orglucose deprivation. Five families have been classified according tomolecular weight: HSP-110, -90, -70, -60 and -28. HSPs deliverimmunogenic peptides through the cross-presentation pathway in antigenpresenting cells (APCs) such as macrophages and dendritic cells (DCs),leading to T cell activation. HSPs function as chaperone carriers oftumor-associated antigenic peptides forming complexes able to inducetumor-specific immunity. Upon release from dying tumor cells, theHSP-antigen complexes are taken up by antigen-presenting cells (APCs)wherein the antigens are processed into peptides that bind MHC class Iand class II molecules leading to the activation of anti-tumor CD8+ andCD4+ T cells. The immunity elicited by HSP complexes derived from tumorpreparations is specifically directed against the unique antigenicpeptide repertoire expressed by the cancer of each subject. Therefore,in one embodiment, the present invention relates to (a) an antibodyand/or pharmaceutical composition of the present invention and (b) avaccine for use as a medicament, for example for use in a method for thetreatment of cancer. In one embodiment, the present invention relates toa pharmaceutical composition, kit or kit-of-parts comprising (a) anantibody and/or pharmaceutical composition of the present invention and(b) a vaccine. In one embodiment, the vaccine is a heat shock proteinbased tumor vaccine. In one embodiment, the vaccine is a heat shockprotein based pathogen vaccine.

A heat shock protein peptide complex (HSPPC) is a protein peptidecomplex consisting of a heat shock protein non-covalently complexed withantigenic peptides. HSPPCs elicit both innate and adaptive immuneresponses. In a specific embodiment, the antigenic peptide(s) displaysantigenicity for the cancer being treated. HSPPCs are efficiently seizedby APCs via membrane receptors (mainly CD91) or by binding to Toll-likereceptors. HSPPC internalization results in functional maturation of theAPCs with chemokine and cytokine production leading to activation ofnatural killer cells (NK), monocytes and Th1 and Th-2-mediated immuneresponses. In certain embodiments, HSPPCs used in methods disclosedherein comprise one or more heat shock proteins from the hsp60, hsp70,or hsp90 family of stress proteins complexed with antigenic peptides. Incertain embodiments, HSPPCs comprise hsc70, hsp70, hsp90, hsp110,grp170, gp96, calreticulin, or combinations of two or more thereof.

In a specific embodiment, the heat shock protein peptide complex (HSPPC)comprises recombinant heat shock proteins (e.g., hsp70 or hsc70) or apeptide-binding domain thereof complexed with recombinant antigenicpeptides. Recombinant heat shock proteins can be produced by recombinantDNA technology, for example, using human hsc70 sequence as described inDworniczak and Mirault, Nucleic Acids Res. 15:5181-5197 (1987) andGenBank accession no. P11142 and/or Y00371, each of which isincorporated herein by reference in its entirety. In certainembodiments, Hsp70 sequences are as described in Hunt and Morimoto Proc.Natl. Acad. Sci. U.S.A. 82 (19), 6455-6459 (1985) and GenBank accessionno. P0DMV8 and/or M11717, each of which is incorporated herein byreference in its entirety. Antigenic peptides can also be prepared byrecombinant DNA methods known in the art.

In certain embodiments, the antigenic peptides comprise a modified aminoacid. In certain embodiments, the modified amino acid comprises apost-translational modification. In certain embodiments, the modifiedamino acid comprises a mimetic of a post-translational modification. Incertain embodiments, the modified amino acid is a Tyr, Ser, Thr, Arg,Lys, or His that has been phosphorylated on a side chain hydroxyl oramine. In certain embodiments, the modified amino acid is a mimetic of aTyr, Ser, Thr, Arg, Lys, or His amino acid that has been phosphorylatedon a side chain hydroxyl or amine.

In a specific embodiment, an anti-TIM-3 (e.g., human TIM-3) antibodydisclosed herein is administered to a subject in combination with a heatshock protein peptide complex (HSPPC), e.g., heat shock protein peptidecomplex-96 (HSPPC-96), to treat cancer. HSPPC-96 comprises a 96 kDa heatshock protein (Hsp), gp96, complexed to antigenic peptides. HSPPC-96 isa cancer immunotherapy manufactured from a subject's tumor and containsthe cancer's antigenic “fingerprint.” In certain embodiments, thisfingerprint contains unique antigens that are present only in thatparticular subject's specific cancer cells and injection of the vaccineis intended to stimulate the subject's immune system to recognize andattack any cells with the specific cancer fingerprint. Therefore, in oneembodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention in combination witha heat shock protein peptide complex (HSPPC) for use as a medicamentand/or for use in a method for the treatment of cancer.

In certain embodiments, the HSPPC, e.g., HSPPC-96, is produced from thetumor tissue of a subject. In a specific embodiment, the HSPPC (e.g.,HSPPC-96) is produced from a tumor of the type of cancer or metastasisthereof being treated. In another specific embodiment, the HSPPC (e.g.,HSPPC-96) is autologous to the subject being treated. In certainembodiments, the tumor tissue is non-necrotic tumor tissue. In certainembodiments, at least 1 gram (e.g., at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,or at least 10 grams) of non-necrotic tumor tissue is used to produce avaccine regimen. In certain embodiments, after surgical resection,non-necrotic tumor tissue is frozen prior to use in vaccine preparation.In some embodiments, the HSPPC, e.g., HSPPC-96, is isolated from thetumor tissue by purification techniques, filtered and prepared for aninjectable vaccine. In certain embodiments, a subject is administered6-12 doses of the HSPPC, e.g., HSPCC-96. In such embodiments, the HSPPC,e.g., HSPPC-96, doses may be administered weekly for the first 4 dosesand then biweekly for the 2-8 additional doses.

Further examples of HSPPCs that may be used in accordance with themethods described herein are disclosed in the following patents andpatent applications, all of which are herein incorporated by referencein their entireties: U.S. Pat. Nos. 6,391,306, 6,383,492, 6,403,095,6,410,026, 6,436,404, 6,447,780, 6,447,781 and 6,610,659.

In certain embodiments, an anti-TIM-3 antibody disclosed herein isadministered to a subject in combination with an adjuvant. Variousadjuvants can be used depending on the treatment context. Non-limitingexamples of appropriate adjuvants include, but not limited to, CompleteFreund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), MONTANIDE™ISA (incomplete Seppic adjuvant), the RIBI ADJUVANT SYSTEM (RAS)®(stable oil-in-water emulsion), TITERMAX® (block copolymer CRL-8941,squalene, a metabolizable oil, and a microparticulate stabilizer),muramyl peptides, SYNTEX ADJUVANT FORMULATIONS (SAF)® (microfluidizedoil-in-water emulsion adjuvant), alum (aluminum hydroxide and/oraluminum phosphate), aluminum salt adjuvants, GERBU ADJUVANTS® (GERBUBiochemicals GmbH) (muramyl glycopeptide and solid ultrafiltrableparticles of slowly biodegradable lipids), nitrocellulose absorbedantigen, encapsulated or entrapped antigen, 3 De-O-acylatedmonophosphoryl lipid A (3 D-MPL), immunostimulatory oligonucleotides,toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands,stimulator of interferon genes (STING) agonists, immuno-stimulatingcomplexes such as saponins, QUIL-A® (saponin adjuvant), QS-21, QS-7,ISCOMATRIX® (saponin adjuvant), and others. Other adjuvants include CpGoligonucleotides and double stranded RNA molecules, such as poly(A) andpoly(U). Combinations of the above adjuvants may also be used. See,e.g., U.S. Pat. Nos. 6,645,495; 7,029,678; and 7,858,589, all of whichare incorporated herein by reference in their entireties. In oneembodiment, the adjuvant used herein is QS-21 STIMUILON® (saponinadjuvant).

In certain embodiments, an anti-TIM-3 antibody disclosed herein isadministered to a subject in combination with an additional therapeuticagent comprising a TCR. In certain embodiments, the additionaltherapeutic agent is a soluble TCR. In certain embodiments, theadditional therapeutic agent is a cell expressing a TCR. Therefore, inone embodiment, the present invention relates to an antibody and/orpharmaceutical composition of the present invention in combination withan additional therapeutic agent comprising a TCR for use as a medicamentand/or for use in a method for the treatment of cancer.

In certain embodiments, an anti-TIM-3 antibody disclosed herein isadministered to a subject in combination with a cell expressing achimeric antigen receptor (CAR). In certain embodiments, the cell is a Tcell.

In certain embodiments, an anti-TIM-3 antibody disclosed herein isadministered to a subject in combination with a TCR mimic antibody. Incertain embodiments, the TCR mimic antibody is an antibody thatspecifically binds to a peptide-MHC complex. For non-limiting examplesof TCR mimic antibodies, see, e.g., U.S. Pat. No. 9,074,000 and U.S.Publication Nos. US 2009/0304679 A1 and US 2014/0134191 A1, all of whichare incorporated herein by reference in their entireties.

The anti-TIM-3 (e.g., human TIM-3) antibody and the additionaltherapeutic agent (e.g., chemotherapeutic, radiotherapeutic, checkpointtargeting agent, IDO inhibitor, vaccine, adjuvant, a soluble TCR, a cellexpressing a TCR, a cell expressing a chimeric antigen receptor, and/ora TCR mimic antibody) can be administered separately, sequentially orconcurrently as separate dosage forms. In one embodiment, an anti-TIM-3(e.g., human TIM-3) antibody is administered parenterally, and an IDOinhibitor is administered orally.

An antibody or pharmaceutical composition described herein may bedelivered to a subject by a variety of routes. These include, but arenot limited to, parenteral, intranasal, intratracheal, oral,intradermal, topical, intramuscular, intraperitoneal, transdermal,intravenous, intrathecal, intratumoral, conjunctival, intra-arterial,and subcutaneous routes. In certain embodiments, the antibody orpharmaceutical composition is delivered intravenously. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent for use as aspray. In certain embodiments, the antibody or pharmaceuticalcomposition described herein is delivered subcutaneously orintravenously. In certain embodiments, the antibody or pharmaceuticalcomposition described herein is delivered intra-arterially. In certainembodiments, the antibody or pharmaceutical composition described hereinis delivered intratumorally. In certain embodiments, the antibody orpharmaceutical composition described herein is delivered into a tumordraining lymph node.

The amount of an antibody or composition which will be effective in thetreatment and/or prevention of a condition will depend on the nature ofthe disease, and can be determined by standard clinical techniques.

The precise dose to be employed in a composition will also depend on theroute of administration, and the seriousness of the infection or diseasecaused by it, and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedoses may also vary depending upon means of administration, target site,physiological state of the patient (including age, body weight andhealth), whether the patient is human or an animal, other medicationsadministered, or whether treatment is prophylactic or therapeutic.Usually, the patient is a human but non-human mammals includingtransgenic mammals can also be treated. Treatment dosages are optimallytitrated to optimize safety and efficacy.

An anti-TIM-3 (e.g., human TIM-3) antibody described herein can also beused to assay TIM-3 (e.g., human TIM-3) protein levels in a biologicalsample using classical immunohistological methods known to those ofskill in the art, including immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA), immunoprecipitation, or Western blotting.Suitable antibody assay labels are known in the art and include enzymelabels, such as, glucose oxidase; radioisotopes, such as iodine (¹²⁵I,¹²¹I) carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In), andtechnetium (⁹⁹Tc); luminescent labels, such as luminol; and fluorescentlabels, such as fluorescein and rhodamine, and biotin. Such labels canbe used to label an antibody described herein. Alternatively, a secondantibody that recognizes an anti-TIM-3 (e.g., human TIM-3) antibodydescribed herein can be labeled and used in combination with ananti-TIM-3 (e.g., human TIM-3) antibody to detect TIM-3 (e.g., humanTIM-3) protein levels. Therefore, in one embodiment, the presentinvention relates to the use of an antibody of the present invention forin vitro detection of TIM-3 (e.g., human TIM-3) protein in a biologicalsample. In a further embodiment, the present invention relates to theuse of an anti-TIM-3 antibody of the invention, for assaying and/ordetecting TIM-3 (e.g., human TIM-3) protein levels in a biologicalsample in vitro, optionally wherein the anti-TIM-3 antibody isconjugated to a radionuclide or detectable label, and/or carries a labeldescribed herein, and/or wherein an immunohistological method is used.

Assaying for the expression level of TIM-3 (e.g., human TIM-3) proteinis intended to include qualitatively or quantitatively measuring orestimating the level of TIM-3 (e.g., human TIM-3) protein in a firstbiological sample either directly (e.g., by determining or estimatingabsolute protein level) or relatively (e.g., by comparing to the diseaseassociated protein level in a second biological sample). TIM-3 (e.g.,human TIM-3) polypeptide expression level in the first biological samplecan be measured or estimated and compared to a standard TIM-3 (e.g.,human TIM-3) protein level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” TIM-3 (e.g., human TIM-3) polypeptide level is known, it canbe used repeatedly as a standard for comparison. Therefore, in a furtherembodiment, the present invention relates to an in vitro method forassaying and/or detecting TIM-3 protein levels, for example human TIM-3protein levels, in a biological sample, comprising qualitatively orquantitatively measuring or estimating the level of TIM-3 protein, forexample of human TIM-3 protein, in a biological sample, by animmunohistological method.

As used herein, the term “biological sample” refers to any biologicalsample obtained from a subject, cell line, tissue, or other source ofcells potentially expressing TIM-3 (e.g., human TIM-3). Methods forobtaining tissue biopsies and body fluids from animals (e.g., humans)are well known in the art. Biological samples include peripheralmononuclear blood cells.

An anti-TIM-3 (e.g., human TIM-3) antibody described herein can be usedfor prognostic, diagnostic, monitoring and screening applications,including in vitro and in vivo applications well known and standard tothe skilled artisan and based on the present description. Prognostic,diagnostic, monitoring and screening assays and kits for in vitroassessment and evaluation of immune system status and/or immune responsemay be utilized to predict, diagnose and monitor to evaluate patientsamples including those known to have or suspected of having an immunesystem-dysfunction or with regard to an anticipated or desired immunesystem response, antigen response or vaccine response. The assessmentand evaluation of immune system status and/or immune response is alsouseful in determining the suitability of a patient for a clinical trialof a drug or for the administration of a particular chemotherapeuticagent, a radiotherapeutic agent, or an antibody, including combinationsthereof, versus a different agent or antibody. This type of prognosticand diagnostic monitoring and assessment is already in practiceutilizing antibodies against the HER2 protein in breast cancer(HERCEPTEST, Dako (immunohistochemical assay for determination of HER2protein)) where the assay is also used to evaluate patients for antibodytherapy using HERCEPTIN® (trastuzumab). In vivo applications includedirected cell therapy and immune system modulation and radio imaging ofimmune responses. Therefore, in one embodiment, the present inventionrelates to an anti-TIM-3 antibody and/or pharmaceutical composition ofthe present invention for use as a diagnostic. In one embodiment, thepresent invention relates to an anti-TIM-3 antibody and/orpharmaceutical composition of the present invention for use in a methodfor the prediction, diagnosis and/or monitoring of a subject having orsuspected to have an immune system-dysfunction and/or with regard to ananticipated or desired immune system response, antigen response orvaccine response. In another embodiment, the present invention relatesto the use of anti-TIM-3 antibody of the invention, for predicting,diagnosing and/or monitoring of a subject having or suspected to have animmune system-dysfunction and/or with regard to an anticipated ordesired immune system response, antigen response or vaccine response byassaying and/or detecting human TIM-3 protein levels in a biologicalsample of the subject in vitro.

In one embodiment, an anti-TIM-3 (e.g., human TIM-3) antibody can beused in immunohistochemistry of biopsy samples. In one embodiment, themethod is an in vitro method. In another embodiment, an anti-TIM-3(e.g., human TIM-3) antibody can be used to detect levels of TIM-3(e.g., human TIM-3), or levels of cells which contain TIM-3 (e.g., humanTIM-3) on their membrane surface, which levels can then be linked tocertain disease symptoms. Anti-TIM-3 (e.g., human TIM-3) antibodiesdescribed herein may carry a detectable or functional label and/or maybe conjugated to a radionuclide or detectable label. When fluorescencelabels are used, currently available microscopy andfluorescence-activated cell sorter analysis (FACS) or combination ofboth methods procedures known in the art may be utilized to identify andto quantitate the specific binding members. Anti-TIM-3 (e.g., humanTIM-3) antibodies described herein may carry or may be conjugated to afluorescence label. Exemplary fluorescence labels include, for example,reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein andTexas red, ALEXA FLUOR® dyes, Cyanine dyes and DYLIGHT® Fluorescentdyes. An anti-TIM-3 (e.g., human TIM-3) antibody may carry or may beconjugated to a radioactive label or radionuclide, such as the isotopes³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁹⁰Y, ⁹⁹Tc, ¹¹¹, ¹¹⁷Lu,¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁹⁸Au, ²¹¹At, ²¹³Bi, ²²⁵Ac and ¹⁸⁶Re. Whenradioactive labels are used, currently available counting proceduresknown in the art may be utilized to identify and quantitate the specificbinding of anti-TIM-3 (e.g., human TIM-3) antibody to TIM-3 (e.g., humanTIM-3). In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques as known in the art. This can be achieved by contacting asample or a control sample with an anti-TIM-3 (e.g., human TIM-3)antibody under conditions that allow for the formation of a complexbetween the antibody and TIM-3 (e.g., human TIM-3). Any complexes formedbetween the antibody and TIM-3 (e.g., human TIM-3) are detected andcompared in the sample and the control. In light of the specific bindingof the antibodies described herein for TIM-3 (e.g., human TIM-3), theantibodies can be used to specifically detect TIM-3 (e.g., human TIM-3)expression on the surface of cells. The antibodies described herein canalso be used to purify TIM-3 (e.g., human TIM-3) via immunoaffinitypurification. Also included herein is an assay system which may beprepared in the form of a test kit, kit or kit-of-parts for thequantitative analysis of the extent of the presence of, for instance,TIM-3 (e.g., human TIM-3) or TIM-3 (e.g., human TIM-3)/TIM-3 (e.g.,human TIM-3) ligand complexes. The system, test kit, kit or kit-of-partsmay comprise a labeled component, e.g., a labeled antibody, and one ormore additional immunochemical reagents.

5.5 Polynucleotides, Vectors and Methods of Producing Anti-TIM-3Antibodies

In another aspect, provided herein are polynucleotides comprising anucleotide sequence encoding an antibody described herein or a fragmentthereof (e.g., a light chain variable region and/or heavy chain variableregion) that specifically binds to a TIM-3 (e.g., human TIM-3) antigen,and vectors, e.g., vectors comprising such polynucleotides forrecombinant expression in host cells (e.g., E. coli and mammaliancells). Provided herein are polynucleotides comprising nucleotidesequences encoding a heavy and/or light chain of any of the antibodiesprovided herein, as well as vectors comprising such polynucleotidesequences, e.g., expression vectors for their efficient expression inhost cells, e.g., mammalian cells.

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular lessthan about 10%) of other material, e.g., cellular material, culturemedium, other nucleic acid molecules, chemical precursors and/or otherchemicals. In a specific embodiment, a nucleic acid molecule(s) encodingan antibody described herein is isolated or purified.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies, which specifically bind to aTIM-3 (e.g., human TIM-3) polypeptide and comprises an amino acidsequence as described herein, as well as antibodies which compete withsuch antibodies for binding to a TIM-3 (e.g., human TIM-3) polypeptide(e.g., in a dose-dependent manner), or which binds to the same epitopeas that of such antibodies.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody described herein. The polynucleotides can comprise nucleotidesequences encoding a light chain comprising the VL FRs and CDRs ofantibodies described herein (see, e.g., Table 1) or nucleotide sequencesencoding a heavy chain comprising the VH FRs and CDRs of antibodiesdescribed herein (see, e.g., Table 1).

Also provided herein are polynucleotides encoding an anti-TIM-3 (e.g.,human TIM-3) antibody that are optimized, e.g., by codon/RNAoptimization, replacement with heterologous signal sequences, andelimination of mRNA instability elements. Methods to generate optimizednucleic acids encoding an anti-TIM-3 (e.g., human TIM-3) antibody or afragment thereof (e.g., light chain, heavy chain, VH domain, or VLdomain) for recombinant expression by introducing codon changes and/oreliminating inhibitory regions in the mRNA can be carried out byadapting the optimization methods described in, e.g., U.S. Pat. Nos.5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly,all of which are herein incorporated by reference in their entireties.For example, potential splice sites and instability elements (e.g., A/Tor A/U rich elements) within the RNA can be mutated without altering theamino acids encoded by the nucleic acid sequences to increase stabilityof the RNA for recombinant expression. The alterations utilize thedegeneracy of the genetic code, e.g., using an alternative codon for anidentical amino acid. In some embodiments, it can be desirable to alterone or more codons to encode a conservative mutation, e.g., a similaramino acid with similar chemical structure and properties and/orfunction as the original amino acid. Such methods can increaseexpression of an anti-TIM-3 (e.g., human TIM-3) antibody or fragmentthereof by at least 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or100 fold or more relative to the expression of an anti-TIM-3 (e.g.,human TIM-3) antibody encoded by polynucleotides that have not beenoptimized.

In certain embodiments, an optimized polynucleotide sequence encoding ananti-TIM-3 (e.g., human TIM-3) antibody described herein or a fragmentthereof (e.g., VL domain and/or VH domain) can hybridize to an antisense(e.g., complementary) polynucleotide of an unoptimized polynucleotidesequence encoding an anti-TIM-3 (e.g., human TIM-3) antibody describedherein or a fragment thereof (e.g., VL domain and/or VH domain). Inspecific embodiments, an optimized nucleotide sequence encoding ananti-TIM-3 (e.g., human TIM-3) antibody described herein or a fragmenthybridizes under high stringency conditions to antisense polynucleotideof an unoptimized polynucleotide sequence encoding an anti-TIM-3 (e.g.,human TIM-3) antibody described herein or a fragment thereof. In aspecific embodiment, an optimized nucleotide sequence encoding ananti-TIM-3 (e.g., human TIM-3) antibody described herein or a fragmentthereof hybridizes under high stringency, intermediate or lowerstringency hybridization conditions to an antisense polynucleotide of anunoptimized nucleotide sequence encoding an anti-TIM-3 (e.g., humanTIM-3) antibody described herein or a fragment thereof. Informationregarding hybridization conditions has been described, see, e.g., U.S.Patent Application Publication No. US 2005/0048549 (e.g., paragraphs72-73), which is herein incorporated by reference in its entirety.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding antibodies described herein, e.g., antibodiesdescribed in Table 1, and modified versions of these antibodies can bedetermined using methods well known in the art, i.e., nucleotide codonsknown to encode particular amino acids are assembled in such a way togenerate a nucleic acid that encodes the antibody. Such a polynucleotideencoding the antibody can be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier G et al., (1994),BioTechniques 17: 242-6, herein incorporated by reference in itsentirety), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described hereincan be generated from nucleic acid from a suitable source (e.g., ahybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the antibody of interest. Such PCR amplification methods canbe used to obtain nucleic acids comprising the sequence encoding thelight chain and/or heavy chain of an antibody. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody. The amplified nucleic acids can be cloned intovectors for expression in host cells and for further cloning, forexample, to generate chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody isnot available, but the sequence of the antibody molecule is known, anucleic acid encoding the immunoglobulin can be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody described herein) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR can then be cloned into replicable cloning vectorsusing any method well known in the art.

DNA encoding anti-TIM-3 (e.g., human TIM-3) antibodies described hereincan be readily isolated and sequenced using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of theanti-TIM-3 (e.g., human TIM-3) antibodies). Hybridoma cells can serve asa source of such DNA. Once isolated, the DNA can be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells(e.g., CHO cells from the CHO GS SYSTEM™ (Lonza)), or myeloma cells thatdo not otherwise produce immunoglobulin protein, to obtain the synthesisof anti-TIM-3 (e.g., human TIM-3) antibodies in the recombinant hostcells.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. In certain embodiments, the vectors for expressing the VH or VLdomains comprise an EF-1a promoter, a secretion signal, a cloning sitefor the variable region, constant domains, and a selection marker suchas neomycin. The VH and VL domains can also be cloned into one vectorexpressing the necessary constant regions. The heavy chain conversionvectors and light chain conversion vectors are then co-transfected intocell lines to generate stable or transient cell lines that expressfull-length antibodies, e.g., IgG, using techniques known to those ofskill in the art.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody described herein. In specificembodiments, polynucleotides described herein hybridize under highstringency, intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH domain and/or VL domain provided herein.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel F M et al., eds., (1989)Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3, which is herein incorporated by reference in itsentirety.

In certain aspects, provided herein are cells (e.g., host cells)expressing (e.g., recombinantly) antibodies described herein whichspecifically bind to TIM-3 (e.g., human TIM-3) and relatedpolynucleotides and expression vectors. Provided herein are vectors(e.g., expression vectors) comprising polynucleotides comprisingnucleotide sequences encoding anti-TIM-3 (e.g., human TIM-3) antibodiesor a fragment for recombinant expression in host cells, preferably inmammalian cells. Also provided herein are host cells comprising suchvectors for recombinantly expressing anti-TIM-3 (e.g., human TIM-3)antibodies described herein (e.g., human or humanized antibody). In aparticular aspect, provided herein are methods for producing an antibodydescribed herein, comprising expressing such antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., afull-length antibody, heavy and/or light chain of an antibody, or asingle chain antibody described herein) that specifically binds to TIM-3(e.g., human TIM-3) involves construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule, heavy and/or light chainof an antibody, or a fragment thereof (e.g., heavy and/or light chainvariable regions) described herein has been obtained, the vector for theproduction of the antibody molecule can be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody or antibody fragment (e.g., light chain or heavy chain)encoding nucleotide sequence are described herein. Methods which arewell known to those skilled in the art can be used to constructexpression vectors containing antibody or antibody fragment (e.g., lightchain or heavy chain) coding sequences and appropriate transcriptionaland translational control signals. These methods include, for example,in vitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Also provided are replicable vectors comprising anucleotide sequence encoding an antibody molecule described herein, aheavy or light chain of an antibody, a heavy or light chain variableregion of an antibody or a fragment thereof, or a heavy or light chainCDR, operably linked to a promoter. Such vectors can, for example,include the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g., International Publication Nos. WO 86/05807and WO 89/01036; and U.S. Pat. No. 5,122,464, which are hereinincorporated by reference in their entireties) and variable regions ofthe antibody can be cloned into such a vector for expression of theentire heavy, the entire light chain, or both the entire heavy and lightchains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody described herein or afragment thereof. Thus, provided herein are host cells containing apolynucleotide encoding an antibody described herein or fragmentsthereof, or a heavy or light chain thereof, or fragment thereof, or asingle chain antibody described herein, operably linked to a promoterfor expression of such sequences in the host cell. In certainembodiments, for the expression of double-chained antibodies, vectorsencoding both the heavy and light chains, individually, can beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below. In certain embodiments, ahost cell contains a vector comprising a polynucleotide encoding boththe heavy chain and light chain of an antibody described herein, or afragment thereof. In specific embodiments, a host cell contains twodifferent vectors, a first vector comprising a polynucleotide encoding aheavy chain or a heavy chain variable region of an antibody describedherein, or a fragment thereof, and a second vector comprising apolynucleotide encoding a light chain or a light chain variable regionof an antibody described herein, or a fragment thereof. In otherembodiments, a first host cell comprises a first vector comprising apolynucleotide encoding a heavy chain or a heavy chain variable regionof an antibody described herein, or a fragment thereof, and a secondhost cell comprises a second vector comprising a polynucleotide encodinga light chain or a light chain variable region of an antibody describedherein. In specific embodiments, a heavy chain/heavy chain variableregion expressed by a first cell associated with a light chain/lightchain variable region of a second cell to form an anti-TIM-3 (e.g.,human TIM-3) antibody described herein. In certain embodiments, providedherein is a population of host cells comprising such first host cell andsuch second host cell.

In a particular embodiment, provided herein is a population of vectorscomprising a first vector comprising a polynucleotide encoding a lightchain/light chain variable region of an anti-TIM-3 (e.g., human TIM-3)antibody described herein, and a second vector comprising apolynucleotide encoding a heavy chain/heavy chain variable region of ananti-TIM-3 (e.g., human TIM-3) antibody described herein.

A variety of host-expression vector systems can be utilized to expressantibody molecules described herein (see, e.g., U.S. Pat. No. 5,807,715,which is herein incorporated by reference in its entirety). Suchhost-expression systems represent vehicles by which the coding sequencesof interest can be produced and subsequently purified, but alsorepresent cells which can, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody moleculedescribed herein in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systems(e.g., green algae such as Chlamydomonas reinhardtii) infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS),CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, andNIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 andBMT10 cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). In aspecific embodiment, cells for expressing antibodies described hereinare CHO cells, for example CHO cells from the CHO GS SYSTEM™ (Lonza). Ina particular embodiment, cells for expressing antibodies describedherein are human cells, e.g., human cell lines. In a specificembodiment, a mammalian expression vector is POPTIVEC™ or pcDNA3.3. In aparticular embodiment, bacterial cells such as Escherichia coli, oreukaryotic cells (e.g., mammalian cells), especially for the expressionof whole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary (CHO) cells, in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking M K & Hofstetter H (1986) Gene 45: 101-5; and Cockett M I etal., (1990) Biotechnology 8(7): 662-7, each of which is hereinincorporated by reference in its entirety). In certain embodiments,antibodies described herein are produced by CHO cells or NS0 cells. In aspecific embodiment, the expression of nucleotide sequences encodingantibodies described herein which specifically bind to TIM-3 (e.g.,human TIM-3) is regulated by a constitutive promoter, inducible promoteror tissue specific promoter.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983)EMBO J 2: 1791-1794), in which the antibody coding sequence can beligated individually into the vector in frame with the lac Z codingregion so that a fusion protein is produced; pIN vectors (Inouye S &Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster S M(1989) J Biol Chem 24: 5503-5509); and the like, all of which are hereinincorporated by reference in their entireties. For example, pGEX vectorscan also be used to express foreign polypeptides as fusion proteins withglutathione 5-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption andbinding to matrix glutathione agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV), for example, can be used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The antibody codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan J &Shenk T (1984) PNAS 81(12): 3655-9, which is herein incorporated byreference in its entirety). Specific initiation signals can also berequired for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression can be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol.153: 516-544, which is herein incorporated by reference in itsentirety).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst,HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 andHsS78Bst cells. In certain embodiments, anti-TIM-3 (e.g., human TIM-3)antibodies described herein are produced in mammalian cells, such as CHOcells.

In a specific embodiment, the antibodies described herein have reducedfucose content or no fucose content. Such antibodies can be producedusing techniques known one skilled in the art. For example, theantibodies can be expressed in cells deficient or lacking the ability ofto fucosylate. In a specific example, cell lines with a knockout of bothalleles of α1,6-fucosyltransferase can be used to produce antibodieswith reduced fucose content. The GS SYSTEM™ with POTELLIGENT® Technology(Lonza) is an example of such a system that can be used to produceantibodies with reduced fucose content.

For long-term, high-yield production of recombinant proteins, stableexpression cells can be generated. For example, cell lines which stablyexpress an anti-TIM-3 (e.g., human TIM-3) antibody described herein canbe engineered. In specific embodiments, a cell provided herein stablyexpresses a light chain/light chain variable region and a heavychain/heavy chain variable region which associate to form an antibodydescribed herein.

In certain aspects, rather than using expression vectors which containviral origins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA/polynucleotide, engineered cells can be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express an anti-TIM-3 (e.g., humanTIM-3) antibody described herein or a fragment thereof. Such engineeredcell lines can be particularly useful in screening and evaluation ofcompositions that interact directly or indirectly with the antibodymolecule.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (SzybalskaE H & Szybalski W (1962) PNAS 48(12): 2026-2034) and adeninephosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23)genes in tk-, hgprt- or aprt-cells, respectively, all of which areherein incorporated by reference in their entireties. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (WiglerM et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance tothe aminoglycoside G-418 (Wu G Y & Wu C H (1991) Biotherapy 3: 87-95;Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan R C(1993) Science 260: 926-932; and Morgan R A & Anderson W F (1993) AnnRev Biochem 62: 191-217; Nabel G J & Felgner P L (1993) TrendsBiotechnol 11(5): 211-5); and hygro, which confers resistance tohygromycin (Santerre R F et al., (1984) Gene 30(1-3): 147-56), all ofwhich are herein incorporated by reference in their entireties. Methodscommonly known in the art of recombinant DNA technology can be routinelyapplied to select the desired recombinant clone and such methods aredescribed, for example, in Ausubel F M et al., (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler M, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, N Y(1990); and in Chapters 12 and 13, Dracopoli N C et al., (eds.), CurrentProtocols in Human Genetics, John Wiley & Sons, N Y (1994);Colbére-Garapin F et al., (1981) J Mol Biol 150: 1-14, all of which areherein incorporated by reference in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington C R & Hentschel CCG, The useof vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987), which is herein incorporated by reference in its entirety).When a marker in the vector system expressing antibody is amplifiable,increase in the level of inhibitor present in culture of host cell willincrease the number of copies of the marker gene. Since the amplifiedregion is associated with the antibody gene, production of the antibodywill also increase (Crouse G F et al., (1983) Mol Cell Biol 3: 257-66,which is herein incorporated by reference in its entirety).

The host cell can be co-transfected with two or more expression vectorsdescribed herein, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides. Thehost cells can be co-transfected with different amounts of the two ormore expression vectors. For example, host cells can be transfected withany one of the following ratios of a first expression vector and asecond expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot N J (1986) Nature322: 562-565; and Köhler G (1980) PNAS 77: 2197-2199, each of which isherein incorporated by reference in its entirety). The coding sequencesfor the heavy and light chains can comprise cDNA or genomic DNA. Theexpression vector can be monocistronic or multicistronic. Amulticistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9,10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotidesequences. For example, a bicistronic nucleic acid construct cancomprise in the following order a promoter, a first gene (e.g., heavychain of an antibody described herein), and a second gene and (e.g.,light chain of an antibody described herein). In such an expressionvector, the transcription of both genes can be driven by the promoter,whereas the translation of the mRNA from the first gene can be by acap-dependent scanning mechanism and the translation of the mRNA fromthe second gene can be by a cap-independent mechanism, e.g., by an IRES.

Once an antibody molecule described herein has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies described herein can be fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an antibody described herein is isolated orpurified.

Generally, an isolated antibody is one that is substantially free ofother antibodies with different antigenic specificities than theisolated antibody. For example, in a particular embodiment, apreparation of an antibody described herein is substantially free ofcellular material and/or chemical precursors. The language“substantially free of cellular material” includes preparations of anantibody in which the antibody is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus, anantibody that is substantially free of cellular material includespreparations of antibody having less than about 30%, 20%, 10%, 5%, 2%,1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referredto herein as a “contaminating protein”) and/or variants of an antibody,for example, different post-translational modified forms of an antibodyor other different versions of an antibody (e.g., antibody fragments).When the antibody is recombinantly produced, it is also generallysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of theprotein preparation. When the antibody is produced by chemicalsynthesis, it is generally substantially free of chemical precursors orother chemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly, such preparations of the antibody have less than about 30%,20%, 10%, or 5% (by dry weight) of chemical precursors or compoundsother than the antibody of interest. In a specific embodiment,antibodies described herein are isolated or purified.

Antibodies or fragments thereof that specifically bind to TIM-3 (e.g.,human TIM-3) can be produced by any method known in the art for thesynthesis of antibodies, for example, by chemical synthesis or byrecombinant expression techniques. The methods described herein employs,unless otherwise indicated, conventional techniques in molecularbiology, microbiology, genetic analysis, recombinant DNA, organicchemistry, biochemistry, PCR, oligonucleotide synthesis andmodification, nucleic acid hybridization, and related fields within theskill of the art. These techniques are described, for example, in thereferences cited herein and are fully explained in the literature. See,e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989),Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel F M et al., Current Protocols in MolecularBiology, John Wiley & Sons (1987 and annual updates); Current Protocolsin Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.)(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press;Eckstein (ed.) (1991) Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: ALaboratory Manual, Cold Spring Harbor Laboratory Press, all of which areherein incorporated by reference in their entireties.

In a specific embodiment, an antibody described herein is an antibody(e.g., recombinant antibody) prepared, expressed, created or isolated byany means that involves creation, e.g., via synthesis, geneticengineering of DNA sequences. In certain embodiments, such antibodycomprises sequences (e.g., DNA sequences or amino acid sequences) thatdo not naturally exist within the antibody germline repertoire of ananimal or mammal (e.g., human) in vivo.

In one aspect, provided herein is a method of making an antibody whichspecifically binds to TIM-3 (e.g., human TIM-3) comprising culturing acell or host cell described herein. In one embodiment, the method isperformed in vitro. In a certain aspect, provided herein is a method ofmaking an antibody which specifically binds to TIM-3 (e.g., human TIM-3)comprising expressing (e.g., recombinantly expressing) the antibodyusing a cell or host cell described herein (e.g., a cell or a host cellcomprising polynucleotides encoding an antibody described herein). In aparticular embodiment, the cell is an isolated cell. In a particularembodiment, the exogenous polynucleotides have been introduced into thecell. In a particular embodiment, the method further comprises the stepof purifying the antibody obtained from the cell or host cell.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York, whichis herein incorporated by reference in its entirety).

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow E & Lane D,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), each of which isherein incorporated by reference in its entirety. The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. For example, monoclonal antibodies can be producedrecombinantly from host cells exogenously expressing an antibodydescribed herein or a fragment thereof, for example, light chain and/orheavy chain of such antibody.

In specific embodiments, a “monoclonal antibody,” as used herein, is anantibody produced by a single cell (e.g., hybridoma or host cellproducing a recombinant antibody), wherein the antibody specificallybinds to TIM-3 (e.g., human TIM-3) as determined, e.g., by ELISA orother antigen-binding or competitive binding assay known in the art orin the examples provided herein. In particular embodiments, a monoclonalantibody can be a chimeric antibody or a humanized antibody. In certainembodiments, a monoclonal antibody is a monovalent antibody ormultivalent (e.g., bivalent) antibody. In particular embodiments, amonoclonal antibody is a monospecific or multispecific antibody (e.g.,bispecific antibody). Monoclonal antibodies described herein can, forexample, be made by the hybridoma method as described in Kohler G &Milstein C (1975) Nature 256: 495, which is herein incorporated byreference in its entirety, or can, e.g., be isolated from phagelibraries using the techniques as described herein, for example. Othermethods for the preparation of clonal cell lines and of monoclonalantibodies expressed thereby are well known in the art (see, forexample, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5thEd., Ausubel F M et al., supra).

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. For example,in the hybridoma method, a mouse or other appropriate host animal, suchas a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunizedto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein (e.g., TIM-3(e.g., human TIM-3)) used for immunization. Alternatively, lymphocytesmay be immunized in vitro. Lymphocytes then are fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding J W (Ed), Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986), herein incorporated byreference in its entirety). Additionally, a RIMMS (repetitiveimmunization multiple sites) technique can be used to immunize an animal(Kilpatrick K E et al., (1997) Hybridoma 16:381-9, herein incorporatedby reference in its entirety).

In some embodiments, mice (or other animals, such as rats, monkeys,donkeys, pigs, sheep, hamster, or dogs) can be immunized with an antigen(e.g., TIM-3 (e.g., human TIM-3)) and once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well-known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theAmerican Type Culture Collection (ATCC®) (Manassas, Va.), to formhybridomas. Hybridomas are selected and cloned by limited dilution. Incertain embodiments, lymph nodes of the immunized mice are harvested andfused with NS0 myeloma cells.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Specific embodiments employ myeloma cells that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asNS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif., USA, and SP-2 or X63-Ag8.653 cells available from the AmericanType Culture Collection, Rockville, Md., USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor D (1984) J Immunol133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), eachof which is herein incorporated by reference in its entirety).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against TIM-3 (e.g., humanTIM-3). The binding specificity of monoclonal antibodies produced byhybridoma cells is determined by methods known in the art, for example,immunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (MA) or enzyme-linked immunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding J W (Ed), Monoclonal Antibodies: Principles and Practice,supra). Suitable culture media for this purpose include, for example,D-MEM or RPMI 1640 medium. In addition, the hybridoma cells may be grownin vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, Protein A-SEPHAROSE® beads, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Antibodies described herein include antibody fragments which recognizespecific TIM-3 (e.g., human TIM-3) and can be generated by any techniqueknown to those of skill in the art. For example, Fab and F(ab′)2fragments described herein can be produced by proteolytic cleavage ofimmunoglobulin molecules, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)2 fragments). A Fab fragmentcorresponds to one of the two identical arms of an antibody molecule andcontains the complete light chain paired with the VH and CH1 domains ofthe heavy chain. A F(ab′)2 fragment contains the two antigen-bindingarms of an antibody molecule linked by disulfide bonds in the hingeregion.

Further, the antibodies described herein can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In particular, DNA sequences encoding VH and VL domains are amplifiedfrom animal cDNA libraries (e.g., human or murine cDNA libraries ofaffected tissues). The DNA encoding the VH and VL domains are recombinedtogether with a scFv linker by PCR and cloned into a phagemid vector.The vector is electroporated in E. coli and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13, and the VH and VL domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to a particular antigencan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Examples ofphage display methods that can be used to make the antibodies describedherein include those disclosed in Brinkman U et al., (1995) J ImmunolMethods 182: 41-50; Ames R S et al., (1995) J Immunol Methods 184:177-186; Kettleborough Calif. et al., (1994) Eur J Immunol 24: 952-958;Persic L et al., (1997) Gene 187: 9-18; Burton D R & Barbas C F (1994)Advan Immunol 57: 191-280; PCT Application No. PCT/GB91/001134;International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and WO 97/13844; andU.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727, 5,733,743 and 5,969,108, all of which are herein incorporatedby reference in their entireties.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceantibody fragments such as Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax R L et al., (1992) BioTechniques12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; andBetter M et al., (1988) Science 240: 1041-1043, all of which are hereinincorporated by reference in their entireties.

In certain embodiments, to generate whole antibodies, PCR primersincluding VH or VL nucleotide sequences, a restriction site, and aflanking sequence to protect the restriction site can be used to amplifythe VH or VL sequences from a template, e.g., scFv clones. Utilizingcloning techniques known to those of skill in the art, the PCR amplifiedVH domains can be cloned into vectors expressing a VH constant region,and the PCR amplified VL domains can be cloned into vectors expressing aVL constant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mouse orrat monoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L(1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J ImmunolMethods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,4,816,397, and 6,331,415, all of which are herein incorporated byreference in their entireties.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Ahumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₃ and IgG₄. Humanized antibodies can be produced using a variety oftechniques known in the art, including but not limited to, CDR-grafting(European Patent No. EP 239400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089),veneering or resurfacing (European Patent Nos. EP 592106 and EP 519596;Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G M et al.,(1994) Prot Engineering 7(6): 805-814; and Roguska M A et al., (1994)PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,International Publication No. WO 93/17105; Tan P et al., (2002) JImmunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al.,(1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996) ProteinEng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23 Supp):5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22; SandhuJ S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) J MolBiol 235(3): 959-73, all of which are herein incorporated by referencein their entireties. See also U.S. Application Publication No. US2005/0042664 A1 (Feb. 24, 2005), which is herein incorporated byreference in its entirety.

Methods for making multispecific (e.g., bispecific antibodies) have beendescribed, see, for example, U.S. Pat. Nos. 7,951,917; 7,183,076;8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713, allof which are herein incorporated by reference in their entireties.

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well known in the art. See RiechmannL & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al.,(2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) JBiotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and InternationalPublication Nos. WO 94/04678, WO 94/25591 and WO 01/44301, all of whichare herein incorporated by reference in their entireties.

Further, antibodies that specifically bind to a TIM-3 (e.g., humanTIM-3) antigen can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” an antigen using techniques well known to thoseskilled in the art. See, e.g., Greenspan N S & Bona C A (1989) FASEB J7(5): 437-444; and Nissinoff A (1991) J Immunol 147(8): 2429-2438, eachof which is herein incorporated by reference in its entirety.

In particular embodiments, an antibody described herein, which binds tothe same epitope of TIM-3 (e.g., human TIM-3) as an anti-TIM-3 (e.g.,human TIM-3) antibody described herein, is a human antibody. Inparticular embodiments, an antibody described herein, whichcompetitively blocks (e.g., in a dose-dependent manner) any one of theantibodies described herein, from binding to TIM-3 (e.g., human TIM-3),is a human antibody. Human antibodies can be produced using any methodknown in the art. For example, transgenic mice which are incapable ofexpressing functional endogenous immunoglobulins, but which can expresshuman immunoglobulin genes, can be used. In particular, the human heavyand light chain immunoglobulin gene complexes can be introduced randomlyor by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion can be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then bred to producehomozygous offspring which express human antibodies. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of an antigen (e.g., TIM-3 (e.g., human TIM-3)). Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93,herein incorporated by reference in its entirety. For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., International Publication Nos. WO 98/24893, WO 96/34096 and WO96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318 and 5,939,598, all of which are hereinincorporated by reference in their entireties. Examples of mice capableof producing human antibodies include the XENOMOUSE® (Abgenix, Inc.;U.S. Pat. Nos. 6,075,181 and 6,150,184), the HUAB-MOUSE™ (Mederex,Inc./Gen Pharm; U.S. Pat. Nos. 5,545,806 and 5,569,825), the TRANSCHROMOMOUSE™ (Kirin) and the KM MOUSE™ (Medarex/Kirin), all of which areherein incorporated by reference in their entireties.

Human antibodies which specifically bind to TIM-3 (e.g., human TIM-3)can be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887,4,716,111, and 5,885,793; and International Publication Nos. WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741, all of which are herein incorporated byreference in their entireties.

In some embodiments, human antibodies can be produced using mouse-humanhybridomas. For example, human peripheral blood lymphocytes transformedwith Epstein-Barr virus (EBV) can be fused with mouse myeloma cells toproduce mouse-human hybridomas secreting human monoclonal antibodies,and these mouse-human hybridomas can be screened to determine ones whichsecrete human monoclonal antibodies that specifically bind to a targetantigen (e.g., TIM-3 (e.g., human TIM-3)). Such methods are known andare described in the art, see, e.g., Shinmoto H et al., (2004)Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31, each of which is herein incorporated by reference in itsentirety.

5.6 Kits

Also provided, are kits comprising one or more antibodies describedherein, or pharmaceutical composition or conjugates thereof. In aspecific embodiment, provided herein is a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions described herein, such asone or more antibodies provided herein. In some embodiments, the kitscontain a pharmaceutical composition described herein and anyprophylactic or therapeutic agent, such as those described herein. Incertain embodiments, the kits may contain a T cell mitogen, such as,e.g., phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA),or a TCR complex stimulating antibody, such as an anti-CD3 antibody andanti-CD28 antibody. Optionally associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

Also provided, are kits that can be used in the above methods. In oneembodiment, a kit comprises an antibody described herein, preferably apurified antibody, in one or more containers. In a specific embodiment,kits described herein contain a substantially isolated TIM-3 (e.g.,human TIM-3) antigen as a control. In another specific embodiment, thekits described herein further comprise a control antibody which does notreact with a TIM-3 (e.g., human TIM-3) antigen. In another specificembodiment, kits described herein contain one or more elements fordetecting the binding of an antibody to a TIM-3 (e.g., human TIM-3)antigen (e.g., the antibody can be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody can be conjugated to a detectablesubstrate). In specific embodiments, a kit provided herein can include arecombinantly produced or chemically synthesized TIM-3 (e.g., humanTIM-3) antigen. The TIM-3 (e.g., human TIM-3) antigen provided in thekit can also be attached to a solid support. In a more specificembodiment, the detecting means of the above described kit includes asolid support to which a TIM-3 (e.g., human TIM-3) antigen is attached.Such a kit can also include a non-attached reporter-labeled anti-humanantibody or anti-mouse/rat antibody. In this embodiment, binding of theantibody to the TIM-3 (e.g., human TIM-3) antigen can be detected bybinding of the said reporter-labeled antibody. In one embodiment, thepresent invention relates to the use of a kit of the present inventionfor in vitro assaying and/or detecting TIM-3 antigen (e.g., human TIM-3)in a biological sample.

6. EXAMPLES

The examples in this Section (i.e., Section 6) are offered by way ofillustration, and not by way of limitation.

6.1 Example 1: Generation and Characterization of Novel AntibodiesAgainst Human TIM-3

This example describes the generation and characterization of antibodiesthat bind to human T cell immunoglobulin and mucin domain-3 (TIM-3). Inparticular, this example describes the generation of human antibodiesthat specifically bind to human TIM-3 and inhibit the function of humanTIM-3.

In some of the studies described below, the activity of the anti-TIM-3antibodies of this invention was compared with that of referenceanti-TIM-3 antibody pab1944w or Hum11. The antibody pab1944w wasgenerated based on the variable regions of the antibody 8213 HV0LV0provided in U.S. Pat. No. 8,552,156 (herein incorporated by reference inits entirety). The sequences of pab1944w are shown in Table 7. Theantibody pab1944w was expressed as an IgG₁ antibody comprising a N297Amutation in the Fc region, numbered according to the EU numberingsystem. The antibody Hum11 was generated based on the variable regionsof the antibody ABTIM3-hum11 provided in U.S. Patent Publication No. US2015/0218274 (herein incorporated by reference in its entirety). Thesequences of Hum11 are shown in Table 7. The antibody Hum11 wasexpressed as an IgG₄ antibody comprising a S228P mutation in the Fcregion, numbered according to the EU numbering system.

TABLE 7 Sequences of reference anti-TIM-3 antibodies. SEQ ID NO:Description Amino acid sequence 80 pab1944w VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVR QAPGQGLEWMGEINPSNGR TNYNEKFKTRVTITADTSTSTAYMELSSLRSEDTAVYY CARGYYLYFDYWGQGTLVT VSS 81 pab1944w VLDIQMTQSPSSLSASVGDRV TITCHASQGIRINIGWYQQ KPGKAPKLLIYHGTNLEDGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCVQYGQFP WTFGQGTKLEIK 89 pab1944wQVQLVQSGAEVKKPGASVK (IgG₁ VSCKASGYTFTSYWMHWVR N297A) QAPGQGLEWMGEINPSNGRfull TNYNEKFKTRVTITADTST length STAYMELSSLRSEDTAVYY heavyCARGYYLYFDYWGQGTLVT chain VSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYASTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQ KSLSLSPG 90pab1944w DIQMTQSPSSLSASVGDRV (IgG₁ TITCHASQGIRINIGWYQQ N297A)KPGKAPKLLIYHGTNLEDG full VPSRFSGSGSGTDFTLTIS length SLQPEDFATYYCVQYGQFPlight WTFGQGTKLEDCRTVAAPS chain VFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSF NRGEC 82Hum11 VH QVQLVQSGAEVKKPGSSVK VSCKASGYTFTSYNMHWVR QAPGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKST STVYMELSSLRSEDTAVYY CARVGGAFPMDYWGQGTTV TVSS 83Hum11 VL AIQLTQSPSSLSASVGDRV TITCRASESVEYYGTSLMQ WYQQKPGKAPKLLIYAASNVESGVPSRFSGSGSGTDFT LTISSLQPEDFATYFCQQS RKDPSTFGGGTKVEIK 91 Hum11 (IgG₄QVQLVQSGAEVKKPGSSVK S228P VSCKASGYTFTSYNMHWVR full QAPGQGLEWMGDIYPGNGDlength TSYNQKFKGRVTITADKST heavy STVYMELSSLRSEDTAVYY chainCARVGGAFPMDYWGQGTTV TVSSASTKGPSVFPLAPCS RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVP SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQK SLSLSLG 92Hum11 AIQLTQSPSSLSASVGDRV (IgG₄ TITCRASESVEYYGTSLMQ S228P)WYQQKPGKAPKLLIYAASN full VESGVPSRFSGSGSGTDFT length LTISSLQPEDFATYFCQQSlight RKDPSTFGGGTKVEIKRTV chain AAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPV TKSFNRGEC

6.1.1 Generation of Anti-TIM-3 Antibodies Using RETROCYTE DISPLAY™Technology

The generation of a RETROCYTE DISPLAY™ antibody library is describedherein.

For the generation of library inserts, total RNA was extracted viaphenol/chloroform from FACS sorted CD19 positive human B lymphocytes.The total RNA was used for first-strand cDNA synthesis using REVERTAID™First Strand cDNA Synthesis Kit from Fermentas (Catalog number (Cat #)K1621 and K1622). Antibody variable regions were amplified from the cDNAby PCR and cloned into retroviral expression vectors (pCMA). Theseconstructs were subsequently used to transduce murine preB cells toexpress antibodies on the surface using RETROCYTE DISPLAY™ technology.

The RETROCYTE DISPLAY™ antibody library generated as described above wasscreened against recombinant human TIM-3 and recombinant cynomolgusTIM-3, leading to the identification of two antibodies, designated aspab2085 and pab2088. The sequence information of the variable regions ofpab2085 and pab2088 is summarized in Table 4. The antibodies pab2085 andpab2088 were expressed as IgG₁ antibodies and analyzed in the assaysdescribed below.

6.1.2 Binding of Anti-TIM-3 Antibodies to TIM-3-Expressing Cells

The antibodies pab2085 and pab2088 were tested for binding toTIM-3-expressing cells using flow cytometry. Briefly, wild type murine1624-5 cells or 1624-5 cells engineered to express human TIM-3 wereincubated with mouse BD FC BLOCK′ (Pharmingen, Cat #553142) to reducenon-specific binding. After washing, the cells were stained with ananti-TIM-3 antibody or an isotype control antibody and analyzed usingFACSCALIBUR™ cell sorter (BD Biosciences). Both pab2085 and pab2088exhibited binding to 1624-5 cells expressing human TIM-3 but not wildtype 1624-5 cells (FIG. 1).

6.1.3 Selectivity Assay for Anti-TIM-3 Antibodies

The selectivity of pab2085 and pab2088 for TIM-3 was assessed againstfamily members TIM-1 and TIM-4 using suspension array technology.LUMINEX® microspheres were coupled with recombinant human TIM-3 Fc (R&DSystems, Cat #2365-TM), recombinant human TIM-3 His (Sino Biological,Cat #10390-H08H), recombinant cynomolgus TIM-3 Fc (R&D Systems, Cat#7914-TM), recombinant human TIM-1 His (R&D Systems, Cat #1750-TM), orrecombinant human TIM-4 His (R&D, Cat #2929-TM), via amine coupling withthe COOH bead surface. Purified pab2085, pab2088, and an IgG₁ isotypecontrol antibody were diluted in assay buffer (Roche, Cat #11112589001)to 10 ng/ml, 100 ng/ml, and 1000 ng/ml. Each dilution (25 μl) wasincubated in the dark (20° C., 650 rpm) with 1500 LUMINEX® microspheresin 5 μl assay buffer for 1 hour in 96 half-well filter plates(Millipore, Cat #MABVN1250). Standard curves were generated usingduplicates of 25 μl of a human IgG₁ kappa standard (Sigma, Cat #15154)with 1:3 dilution series (0.08-540 ng/ml). Detection was carried outusing 60 μl of goat anti-human IgG F(ab)₂ labeled with R-PE (2.5 μg/ml;Jackson ImmunoResearch, Cat #109-116-097) and another hour of incubationtime (20° C., 650 rpm). Plates were analyzed using a LUMINEX® 200 system(Millipore). A total of 100 beads were counted per well in a 48 μlsample volume. PE MFI values were used to determine specific ornon-specific binding to the recombinant proteins mentioned above.

Both pab2085 (FIG. 2A) and pab2088 (FIG. 2B) showed specific binding tohuman and cynomolgous TIM-3, and no significant binding to TIM-1 orTIM-4 was observed at the concentrations tested.

6.1.4 Optimization of Anti-TIM-3 Antibodies Using RETROCYTE DISPLAY™Technology

The antibodies pab2085 and pab2088 share the same heavy chain. To obtainadditional anti-TIM-3 antibodies, a heavy chain RETROCYTE DISPLAY™antibody sub-library was generated based on the heavy chain of pab2085and pab2088 and combined with a more diverse light chain library. Thisnew RETROCYTE DISPLAY™ antibody library was further screened againstrecombinant human TIM-3 and recombinant cynomolgus TIM-3, leading to theidentification of light-chain optimized variants: pab2173, pab2174,pab2175, pab2176, pab2177, pab2178, pab2179, pab2180, pab2181, pab2182,pab2183, pab2184, pab2185, pab2186, pab2187, pab2188, pab2189, pab2190,pab2191, and pab2192. The sequence information of the variable regionsof these light-chain optimized variants is listed in Table 4. Thelight-chain optimized variants were expressed as antibodies containing awild type IgG₁ Fc region or an IgG₁ variant Fc region. This IgG₁ variantFc region does not affect effector functions of the Fc region.

The light-chain optimized antibody pab2188 is an antibody containing aT109S substitution (i.e., substitution of threonine with serine atposition 109 relative to the wild type sequence), numbered according toKabat, in the light chain constant domain, which facilitates the cloningof the variable region in frame to the constant region. This mutation isa conservative modification that does not affect antibody binding orfunction. The wild type counterpart, designated as pab2188w, whichcontains a threonine at position 109 of the light chain, numberedaccording to Kabat, was also generated. The antibody pab2188w wasexpressed as an antibody containing an IgG₁ N297A Fc region.

6.1.5 Binding of anti-TIM-3 antibodies to TIM-3-expressing cells

The light-chain optimized variants were assessed for binding to cellsexpressing human or cynomolgus TIM-3 in a flow cytometry assay similarto the one described above. All the variants exhibited binding to murine1624-5 cells engineered to express human TIM-3 (FIGS. 3A and 3B) orcynomolgus TIM-3 (FIGS. 3C and 3D), but not wild type murine 1624-5cells (data not shown).

The binding of the light-chain optimized variants to primary human Tcells was compared with that of the parental antibody pab2085. Briefly,peripheral blood mononuclear cells (PBMCs) isolated via ficoll gradientfrom healthy donor buffy coats (Research Blood Components, LLC) wereenriched for untouched pan-T cells using magnetic-based separation(Miltenyi Biotec). The enriched population of T lymphocytes was thenactivated with plate-bound anti-CD3 antibody (SP34, 3 μg/ml) and solubleanti-CD28 antibody (CD28.1, 2 μg/ml) for 3 days in RPMI media,supplemented with 10% heat-inactivated FBS, at 37° C. and 5% CO₂.Following activation, cells were incubated with human Fc-receptor blockfor 15 minutes at room temperature to reduce non-specific binding (FcRblock, Biolegend). Anti-TIM-3 or IgG isotype control antibodies(12-point dose titration, 10,000 ng/ml to 0.06 ng/ml) were added toindividual samples and incubated for 30 minutes at 4° C. Samples werewashed two times and an antibody cocktail, containing FITC-conjugatedanti-kappa antibody as well as anti-CD3 (BV711, OKT3), anti-CD4 (BV605,OKT4) and anti-CD8a (PE, RPA-T8, all at 2.5 μg/ml, was diluted in buffer(PBS, 2 mM EDTA, 0.5% BSA, pH 7.2), added to each sample and incubatedfor 30 minutes at 4° C. Samples were washed two times and analyzed usingthe LSRFORTESSA™ flow cytometer (BD Biosciences). Flow cytometry plotswere analyzed using a combination of FACSDIVA™ and WEHI Weasel software.

As shown in FIG. 4, all the light-chain optimized variants tested inthis study showed stronger binding to activated human CD8+ T cells thanthe parental antibody pab2085 did.

Next, the anti-TIM-3 antibody pab2188 was examined for its binding toprimary cynomolgus cells. Cryopreserved PBMCs isolated from cynomolgusmonkeys (Worldwide Primates, Inc.) were thawed, washed and thensubjected to flow cytometric analysis. Prior to antibody incubation, thecells were treated with 10% cynomolgus monkey serum (Abcam) for 15minutes at room temperature to reduce non-specific binding. Anti-TIM-3or IgG isotype control antibodies (10-point dose titration, 20,000 ng/mlto 0.6 ng/ml) were added to individual samples and incubated for 30minutes at 4° C. Samples were washed two times and an antibody cocktail,containing FITC-conjugated anti-kappa antibody as well as anti-CD11b(BV785, M1/70) at 2.5 μg/ml diluted in buffer (PBS, 2 mM EDTA, 0.5% BSA,pH 7.2), was added to each sample and incubated for 30 minutes at 4° C.Samples were washed two times and analyzed using the LSRFORTESSA™ flowcytometer (BD Biosciences). Flow cytometry plots were analyzed using acombination of FACSDIVA™ and WEHI Weasel software.

As shown in FIG. 5, the anti-TIM-3 antibody pab2188 bound primarycynomolgus myeloid cells in a dose-dependent manner.

6.1.6 Ligand Blocking Activity of Anti-TIM-3 Antibodies

Anti-TIM-3 antibodies were tested for their ability to block the bindingof recombinant human or cynomolgus TIM-3 to phosphatidylserine expressedby irradiated WR19L murine lymphoma cells. Anti-TIM-3 or IgG isotypecontrol antibodies (9-point dose titration, 20,000 ng/ml to 70 ng/ml forhuman; or 6-point dose titration, 20,000 ng/ml to 625 ng/ml forcynomolgus monkey) were incubated with recombinant human TIM-3 Fc (R&DSystems, #2365-TM) or recombinant cynomolgus TIM-3 Fc (R&D Systems,#7914-TM) (10,000 ng/ml) prepared in 1× Annexin-V binding buffer (10 mMHepes adjusted to pH 7.4, 140 mM NaCl and 2.5 mM CaCl2) for 30 minutesat room temperature. WR19L cells irradiated at 20 Gy and resuspended in1× Annexin-V binding buffer were added to the anti-TIM-3: TIM-3-Fccocktail at a final density of 1×10⁶ cells/ml and incubated at roomtemperature for 45 minutes. Samples were washed once and an antibodycocktail, containing PE-conjugated anti-Fc antibody (1:100 dilution) aswell as viability stain (Biolegend, NIR channel; 1:1000 dilution)diluted in 1× Annexin-V binding buffer, was added to each sample andincubated for 20 minutes at room temperature. Samples were then washedonce in 1× Annexin-V binding buffer, resuspended in 150 μl buffer (PBS,2 mM EDTA, 0.5% BSA, pH 7.2) and analyzed using the LSRFORTESSA™ flowcytometer (BD Biosciences). Flow cytometry plots were analyzed usingFACSDIVA™ software.

The anti-TIM-3 antibodies pab2085 and pab2188 blocked the binding ofrecombinant human TIM-3 (FIG. 6A) and recombinant cynomolgus TIM-3 (FIG.6B) to phosphatidylserine.

6.1.7 Effect of Anti-TIM-3 Antibodies on Human PBMCs Upon StaphylococcusEnterotoxin A (SEA) Stimulation

The functional activity of the light-chain optimized variants on primaryhuman PBMCs were assessed following Staphylococcus Enterotoxin A (SEA)stimulation. In brief, cryopreserved human PBMCs (Research BloodComponents) were plated at 1×10⁵ cells/well in RPMI1640 supplementedwith NORMOCIN™ Antimicrobial Reagent (Invivogen #ant-nr) and 10%heat-inactivated FBS (GIBCO™, Invitrogen Corporation) in a 96-wellNUNCLON™ delta surface plate (NUNC™). Cells were cultured in thepresence of 5 μg/ml of the anti-PD-1 antibody pembrolizumab (lot7002688300, Myoderm), anti-TIM-3 antibody (10 μg/ml), and the SEAsuperantigen (100 ng/ml, Toxin Technologies) for 6 days at 37° C. and 5%CO₂. Cell-free supernatant was collected and stored at −80° C. untilanalysis. IFNγ levels were determined using an ALPHALISA® immunoassay(Perkin Elmer).

When combined with the anti-PD-1 antibody pembrolizumab, the light-chainoptimized variants enhanced IFNγ production in this primary human PBMCassay (FIG. 7).

The functional activity of pab2188w was analyzed in the SEA stimulationassay using a modified protocol. Cryopreserved human PBMCs (ResearchBlood Components) were plated at 1×10⁵ cells/well in RPMI1640supplemented with NORMOCIN™ Antimicrobial Reagent (Invivogen #ant-nr)and 10% heat-inactivated FBS (GIBCO™, Invitrogen Corporation) in a96-well NUNCLON™ delta surface plate (NUNC™). Cells were cultured in thepresence of 5 μg/ml of the anti-PD-1 antibody pembrolizumab (lot7002688300, Myoderm), anti-TIM-3 antibody (10 μg/ml), and the SEAsuperantigen (100 ng/ml, Toxin Technologies) for 9 days at 37° C. and 5%CO₂. The cells were then washed once and re-stimulated with fresh SEAand antibody for 2 days. Cell-free supernatant was collected and storedat −80° C. until analysis. IFNγ levels were determined using anALPHALISA® immunoassay (Perkin Elmer).

As shown in FIGS. 8A-8F, the anti-TIM-3 antibody pab2188w (IgG₁ N297A),either alone or in combination with the anti-PD-1 antibodypembrolizumab, enhanced IFNγ production in human PBMCs from multipledonors in this SEA stimulation assay.

6.2 Example 2: Optimization of Anti-TIM-3 Antibodies Using CDRMutagenesis

To improve binding affinity, the anti-TIM-3 antibody pab2188w wasmodified using directed mutagenesis of CDR residues of the heavy andlight chain variable regions. Briefly, six Fab phage display librarieswere generated based on the parental antibody pab2188w, each containinga CDRH or a CDRL region modified using NNK degenerate codonrandomization. The Fab phage libraries were subjected to affinity-drivenselections against recombinant human and cynomolgus TIM-3 antigens. Nineclones, designated AM-1, AM-2, AM-3, AM-4, AM-5, AM-6, AM-7, AM-8, andAM-9, were selected based on binding and off-rate measurement. Thesequence information of the variable regions of these nine clones issummarized in Table 4. All of these variants share the light chain ofpab2188w but contain mutations in heavy chain CDR1. AM-1 to AM-9 wereexpressed as full length antibodies containing an IgG₁ N297A Fc regionand analyzed in the experiments described below.

6.2.1 Binding of Anti-TIM-3 Antibodies to TIM-3-Expressing Cells

The binding of antibodies AM-1 to AM-9 to Jurkat cells ectopicallyexpressing human TIM-3 was compared with that of the parental antibodypab2188w in a flow cytometry analysis. As shown in FIG. 9A, all thevariants bound to TIM-3-expressing Jurkat cells and AM-2 and AM-6 showedstronger binding than the parental antibody pab2188w did. The binding ofAM-2 and AM-6 was further analyzed by flow cytometry using Kasumi-3(ATCC® CRL-2725™), a human acute myeloid leukemia cell line endogenouslyexpressing TIM-3 (FIG. 9B), as well as human CD8+ T cells stimulatedwith Staphylococcal Enterotoxin A (SEA) (FIG. 9C) and cynomolgus CD8+ Tcells stimulated with SEA (FIG. 9D). For binding to human CD8+ T cells,human PBMCs isolated via ficoll gradient from healthy donor buffy coats(Research Blood Components, LLC) were activated with SEA (100 ng/ml) for8 days in RPMI media supplemented with 10% heat-inactivated FBS at 37°C. and 5% CO₂. Following activation, cells were incubated with humanFc-receptor block for 15 minutes at room temperature to reducenon-specific binding (FcR block, Biolegend). Anti-TIM-3 or IgG isotypecontrol antibodies (12-point dose titration, 10,000 ng/ml to 0.06 ng/ml)were added to individual samples and incubated for 30 minutes at 4° C.Similarly, for binding to cynomolgus CD8+ T cells, isolated cynomolgusPBMCs were thawed from frozen stocks (Worldwide Primates Inc.) and wereactivated with SEA (100 ng/ml) for five days in RPMI media supplementedwith 10% heat-inactivated FBS at 37° C. and 5% CO₂. Activated cynomolgusmonkey PBMCs were incubated with a combination of human Fc-receptorblock (FcR block, Biolegend) and cynomolgus monkey serum (Abcam) for 15minutes at room temperature to reduce non-specific binding.Phycoerythrin-conjugated AM-2 antibody or isotype control antibody(Biolegend PE-conjugation, 6-point dose titration, 10,000 ng/ml to 41ng/ml) and a cocktail of anti-CD4 antibody (BV605, OKT4) and anti-CD8aantibody (PE, SK1), each at 2.5 μg/ml, was diluted in buffer (PBS, 2 mMEDTA, 0.5% BSA, pH 7.2), added to each sample, and incubated for 30minutes at 4° C. Samples were washed two times and an antibody cocktail,containing FITC-conjugated anti-kappa antibody as well as anti-CD3(BV711, OKT3), anti-CD4 (BV605, OKT4), and anti-CD8a (PE, RPA-T8), allat 2.5 μg/ml, was diluted in buffer (PBS, 2 mM EDTA, 0.5% BSA, pH 7.2),added to each sample, and incubated for 30 minutes at 4° C. Samples werewashed two times and analyzed using the LSRFORTESSA™ flow cytometer (BDBiosciences). Flow cytometry plots were analyzed using a combination ofFACSDIVA™ and WEHI Weasel software. Both AM-2 and AM-6 exhibited bindingto Kasumi-3 cells (FIG. 9B) and activated human CD8+ T cells (FIG. 9C).AM-2 also exhibited binding to activated cynomolgus CD8+ T cells (FIG.9D).

Next, in a similar assay, binding to primary human and cynomolgus CD14+myeloid cells was analyzed by flow cytometry using phycoerythrin(PE)-conjugated pab2188w, AM-2, or an isotype control antibody. Briefly,cryopreserved PBMCs isolated from humans or cynomolgus monkeys(Worldwide Primates, Inc.) were thawed, washed, and then subjected toflow cytometric analysis. Prior to antibody incubation, the cells weretreated with 10% cynomolgus monkey serum (Abcam, Cat #ab155109) for 15minutes at room temperature to reduce non-specific binding.PE-conjugated anti-TIM-3 or IgG isotype control antibodies (12-pointdose titrations, 10,000 ng/ml to 0.05 ng/ml for human PBMCs and 100,000ng/ml to 0.5 ng/ml for cynomolgus monkey PBMCs) were added to individualsamples in an antibody cocktail containing anti-CD14 antibody (APC,M5E2) and ZOMBIE GREEN™ fixable viability marker and then incubated for30 minutes at 4° C. Additional samples were set aside for single staincompensation controls (CD45-FITC, CD45-PE, and CD45-APC; clone MB4-6D6,Miltenyi). Samples were washed two times in buffer and analyzed usingLSRFORTESSA™ flow cytometer (BD Biosciences). Flow cytometry plots wereanalyzed using a combination of FACSDIVA™ and WEHI Weasel software. AM-2showed stronger binding to human (FIG. 9E) and cynomolgus (FIG. 9F)CD14+ myeloid cells than the parental antibody pab2188w did.

6.2.2 Selectivity Assay for Anti-TIM-3 Antibodies

The selectivity of AM-2 and AM-6 for TIM-3 was assessed using suspensionarray technology. LUMINEX® microspheres were coupled with recombinanthuman TIM-3 His (Sino Biological, #10390-H08H), recombinant cynomolgusTIM-3 Fc (Sino Biological, #90312-C02H), recombinant mouse TIM-3 Fc (R&DSystems, #1529-TM), recombinant human TIM-1 His (R&D Systems, #1750-TM),recombinant human TIM-4 His (R&D, #2929-TM), recombinant human OX40 His(Sino Biological, #10481-H08H), recombinant human GITR Fc (R&D Systems,#689-GR), recombinant human DR3 Fc (R&D Systems, #943-D3), andrecombinant human CD137 Fc (in house produced material), via aminecoupling with the COOH bead surface. Purified pab2188w (IgG₁ N297A),AM-2 (IgG₁ N297A), AM-6 (IgG₁ N297A), and an IgG₁ N297A isotype controlantibody were diluted in assay buffer (Roche 11112589001) to a dosetitration from 10000 ng/ml to 0.1 ng/ml. Each dilution (25 μl) wasincubated in the dark (20° C., 650 rpm) with 1500 LUMINEXx microspheresin 5 μl assay buffer for 1 hour in 96 half-well filter plates(Millipore, MABVN1250). Detection was carried out using 60 μl of goatanti-human IgG F(ab)₂ labeled with R-PE (2.5 μg/ml; JIR 109-116-097) andanother hour of incubation time (20° C., 650 rpm). Plates were analyzedusing a LUMINEX® 200 system (Millipore). A total of 100 beads werecounted per well in a 48 μl sample volume. PE MFI values were used todetermine specific or non-specific binding to the recombinant proteins.

The anti-TIM-3 antibodies pab2188w (FIG. 10B), AM-2 (FIG. 10C), and AM-6(FIG. 10D) showed specific binding to human and cynomolgous TIM-3, andno significant binding was detected to mouse TIM-3, human TIM-1, humanTIM-4, human OX40, human GITR, human DR3, or human CD137 at testedconcentrations.

6.2.3 Ligand Blocking Activity of Anti-TIM-3 Antibodies

The anti-TIM-3 antibodies AM-2 and AM-6 were further analyzed for theirability to block the binding of phosphatidylserine to human orcynomolgus TIM-3. Briefly, anti-TIM-3 or IgG isotype control antibodies(10-point dose titration, 40,000 ng/ml to 1000 ng/ml) were incubatedwith recombinant human TIM-3 Fc (R&D Systems, #2365-TM) or recombinantcynomolgus TIM-3 Fc (R&D Systems, #7914-TM) (10,000 ng/ml) prepared in1× Annexin-V binding buffer (10 mM Hepes adjusted to pH 7.4, 140 mM NaCland 2.5 mM CaCl₂)) for 30 minutes at room temperature. WR19L cellsirradiated at 20 Gy and resuspended in 1×Annexin-V binding buffer wereadded to the anti-TIM-3: TIM-3-Fc cocktail at a final density of 1×10⁶cells/ml and incubated at room temperature for 45 minutes. Samples werewashed once and an antibody cocktail, containing PE-conjugated anti-Fcantibody (1:100 dilution) as well as viability stain (Biolegend, NIRchannel; 1:1000 dilution) diluted in 1× Annexin-V binding buffer, wasadded to each sample and incubated for 20 minutes at room temperature.Samples were then washed once in 1×Annexin-V binding buffer and analyzedusing the LSRFORTESSA™ flow cytometer (BD Biosciences). Flow cytometryplots were analyzed using FACSDIVA™ software.

As shown in FIGS. 11A and 11B, the anti-TIM-3 antibodies pab2188w, AM-2,and AM-6 effectively blocked the binding of human or cynomolgus TIM-3 tophosphatidylserine-expressing cells.

6.2.4 Effect of Anti-TIM-3 Antibodies on Human PBMCs Upon StaphylococcusEnterotoxin A (SEA) Stimulation

The functional activity of the variants of pab2188w was analyzed usingprimary human PBMCs stimulated by Staphylococcus Enterotoxin A (SEA).Briefly, cryopreserved human PBMCs (Research Blood Components) wereplated at 1×10⁵ cells/well in RPMI1640 supplemented with NORMOCIN™Antimicrobial Reagent (Invivogen #ant-nr) and 10% heat-inactivated FBS(GIBCO™, Invitrogen Corporation) in 96-well NUNCLON™ delta surfaceplates (NUNC™). Cells were cultured in the presence of 5 μg/ml of theanti-PD-1 antibody pembrolizumab (lot 7002688300, Myoderm), anti-TIM-3antibody (10 μg/ml), and the SEA superantigen (100 ng/ml, ToxinTechnologies) for 9 days at 37° C. and 5% CO₂. The cells were thenwashed once and re-stimulated with fresh SEA and antibody for 2 days.Cell-free supernatant was collected and stored at −80° C. untilanalysis. IFNγ levels were determined using an ALPHALISA® immunoassay(Perkin Elmer).

As shown in FIGS. 12A and 12B, many variants of pab2188w, either aloneor in combination with the anti-PD-1 antibody pembrolizumab, enhancedIFNγ production in human PBMCs from two different donors.

6.2.5 Effect of Anti-TIM-3 Antibodies on Cytokine Production of TumorInfiltrating Lymphocytes

The anti-TIM-3 antibodies were further assessed for their ability tostimulate cytokine production of activated primary tumor infiltratinglymphocytes (TILs), alone or in combination with an anti-PD-1 antibody.Single-cell suspensions from fresh non-small cell lung cancer (NSCLC)(stage II), gallbladder adenocarcinoma (stage IV), or breast cancer(stage II) tumors (UMass Medical School, Worcester, Mass.) were isolatedvia mechanical microdissection. In some cases, depending on the level offibrosis, enzymatic digestion was necessary (Liberase and DNAseI,Roche). Cells were rested at 5×10⁴ cells/well in RPMI1640 supplementedwith NORMOCIN™ Antimicrobial Reagent (Invivogen #ant-nr), recombinanthuman IL-2 (20 U/ml, R&D Systems), and 10% heat-inactivated FBS (GIBCO™,Invitrogen Corporation) in 96-well NUNCLON™ delta surface plates (NUNC™)for 1 day. On the following day, the samples were centrifuged and freshculture media containing the antibodies of interest (anti-TIM-3antibodies at 20 μg/ml and the anti-PD-1 antibody pembrolizumab at 5μg/ml) and anti-CD3/CD28 microbeads (1:1 bead:cell ratio) was added at afinal volume of 100 μl and allowed to incubate for 3 days at 37° C. and5% CO₂. Cell-free supernatant was collected and stored at −80° C. untilanalysis. IFNγ and TNFα levels were determined using an ALPHALISA®immunoassay (Perkin Elmer).

As shown in FIGS. 13A-13F, the anti-TIM-3 antibodies enhanced IFNγ andTNFα production by activated primary TILs from NSCLC, gallbladderadenocarcinoma, or breast cancer tumors.

6.2.6 Internalization of Anti-TIM-3 Antibodies Upon Binding

In this example, internalization of anti-TIM-3 antibodies into cells wasanalyzed. In a first set of experiments, anti-TIM-3 antibodyinternalization was assessed using αHFc-NC-DM1 (anti-human IgG Fcantibody conjugated to maytansinoid DM1 with a non-cleavable linker,Moradec LLC). This secondary antibody drug conjugate αHFc-NC-DM1 bindsto a test antibody (e.g., an anti-TIM-3 antibody) and results in releaseof the cytotoxic payload DM1 into the cytoplasm of the cell uponinternalization. In a second set of experiments, internalization wasevaluated using anti-TIM-3 antibodies pab2188w (IgG₁ N297A) and Hum11(IgG₄ S228P) directly conjugated to monomethyl auristatin E (MMAE). Eachantibody exhibited similar drug-antibody ratios (DAR; Isotypecontrol=3.5, pab2188w=4.0, Hum11=3.0), supporting an equivalent level ofantibody-drug conjugate (ADC) delivery upon internalization. In a thirdset of experiments, internalization was assessed by the subcellularlocalization of a TIM-3 protein labeled with a cell-impermeablefluorescent dye.

Briefly, Kasumi-3 (ATCC® CRL-2725™), an acute myeloid leukemia cell lineendogenously expressing TIM-3, and a Jurkat cell line engineered tooverexpress TIM-3 were plated in white-bottom tissue culture plates at adensity of 2×10⁴ per well. For the first set of experiments using thesecondary antibody drug conjugate αHFc-NC-DM1, an 8-point dose titration(3,333 ng/ml to 1 ng/ml) of either anti-TIM-3 antibody or IgG isotypecontrol antibody in concert with αHFc-NC-DM1 (1:1 with the primaryantibody) was added to the cells at a final volume of 100 μl/well. Thecells were incubated with the primary antibodies and the secondaryantibody drug conjugate at 37° C. and 5% CO₂ for 72 hours.

The anti-TIM-3 antibodies pab2188w (IgG₁ N297A), AM-2 (IgG₁ N297A), andAM-6 (IgG₁ N297A) internalized TIM-3 expressed on Jurkat cells (FIG.14A) and Kasumi-3 cells (FIG. 14B) in the αHFc-NC-DM1 experiments moreeffectively than the reference anti-TIM-3 antibodies Hum11 (IgG₄ S228P)and pab1944w (IgG₁ N297A) did, as evidenced by a greater reduction ofcell survival across a broad range of antibody concentrations.

For the second set of experiments, antibodies pab2188w (IgG1 N297A) andHum11 (ref, IgG4 S228P) were directly conjugated to similarconcentrations of MMAE to account for potential differences in thepropensity of the secondary drug conjugate (αHFc-NC-DM1) to bind thedifferent Fc regions of the antibodies. A 9-point dose titration (6,666ng/ml to 1 ng/ml) of either MMAE-conjugated anti-TIM-3 antibody orMMAE-conjugated IgG isotype control antibody was added to the cells at afinal volume of 100 μl/well. The cells were incubated with theconjugated antibodies at 37° C. and 5% CO₂ for 72 hours. Followingincubation, 90 μl of a preparation of reconstituted CELLTITER-GLO®luminescent cell viability assay (Promega) was added to each well andthe cells were incubated at room temperature for 5 minutes. Theresulting luminescence was recorded using an ENVISION® Plate Reader(Perkin Elmer).

As shown in FIG. 14C, antibody pab2188w (IgG1 N297A) induced a greaterreduction of cell survival than did antibody Hum11 (ref, IgG4 S228P),indicating that the effect observed with the secondary antibody drugconjugate (e.g., as shown in FIG. 14A), was attributable to theinternalization potential of each TIM-3 antibody.

In the third set of experiments, internalization of anti-TIM-3antibodies was analyzed by confocal fluorescence microscopy of livecells. Jurkat cells expressing a HALOTAG® peptide tag-TIM-3 fusionprotein were first incubated with 1 μM Violet Proliferation Dye 450 (BDHORIZON™) for 30 minutes at 37° C. and 5% CO₂. After incubation, cellswere washed in PBS and resuspended in cell culture media. To detect theextracellular domain of TIM-3, the Jurkat HALOTAG® peptide tag-TIM-3expressing cells were stained with a membrane-impermeable HALOTAG® ALEXAFLUOR® 488 fluorescent ligand (Promega, 1 μM) for 15 minutes at 37° C.and 5% CO₂. Cells were then resuspended in fresh culture media andplated in a 384-well microscopy plate (15,000 cells/well) with eitheranti-TIM-3 antibody AM-2 (IgG₁ N297A) or an isotype control (eachantibody at 10 μg/ml). Live images were collected using an IMAGEXPRESS®Micro Confocal High-Content microscope (Molecular Devices) underenvironmental control (37° C. and 5% CO₂) and images were acquired every30 minutes over a course of 3.5 hours. Image analysis was performedusing METAXPRESS® analysis software (Molecular Devices). Jurkat cellswere identified from the DAPI channel (Violet Proliferation Dye 450 (BDHORIZON™)) and the amount of internalized TIM-3 signal was quantifiedper cell from the FITC channel (HALOTAG® ALEXA FLUOR® 488 fluorescentligand).

As shown in FIG. 15, an increase in TIM-3 internalization over time wasobserved for cells incubated with anti-TIM-3 antibody AM-2 relative tocells incubated with isotype control antibody. In particular, after 3.5hours, AM-2 antibody treatment resulted in twice the percentage ofTIM-3-positive cells showing TIM-3 internalization compared toTIM-3-positive cells treated with the isotype control antibody (i.e.,15.1% internalization versus 7.2% internalization, respectively).Further, the internalization signal observed for AM-2 antibody-treatedcells was significantly higher at 3.5 hours than that of cells treatedwith isotype control antibody (p=0.00027, one-tailed T test). There wasno statistically significant difference at the 0-hour time point(p=0.91, one-tailed T-test).

6.3 Example 3: Epitope Mapping of Anti-TIM-3 Antibodies

In this example, the epitope of the anti-TIM-3 antibodies pab2188 (IgG₁variant), pab2187 (IgG₁ variant), and AM-2 (IgG₁ N297A) wascharacterized.

6.3.1 Epitope mapping of anti-TIM-3 antibodies using alanine scanning

The binding characteristics of the anti-TIM-3 antibodies pab2188 (IgG₁variant) and pab2187 (IgG₁ variant) were assessed by alanine scanning.Briefly, the QUIKCHANGE® HT Protein Engineering System from AgilentTechnologies (Cat #G5901A) was used to generate human TIM-3 mutants withalanine substitutions in the extracellular domain. The human TIM-3mutants were expressed on the surface of the murine 1624-5 pre-B cellsusing retroviral transduction. The transduction efficiency or thepercentage of cells expressing human TIM-3 was kept below 5% to ensurethat most cells did not express two or more different TIM-3 mutants.

Cells expressing correctly folded human TIM-3 mutants, as evidenced bybinding to a polyclonal anti-TIM-3 antibody (R&D Systems, Cat #AF2365)in flow cytometry, were further selected for a sub-population thatexpressed human TIM-3 mutants that did not bind the monoclonalanti-TIM-3 antibody pab2188 (IgG₁ variant) or pab2187 (IgG₁ variant).Cells that exhibited specific antibody binding were separated from thenon-binding cell population by preparative, high-speed FACS (FACSARIA™II, BD Biosciences). Antibody reactive or non-reactive cell pools wereexpanded again in tissue culture and cycles of antibody-directed cellsorting and tissue culture expansion were repeated until a clearlydetectable anti-TIM-3 antibody (pab2188 (IgG₁ variant) or pab2187 (IgG₁variant)) non-reactive cell population was obtained. This anti-TIM-3antibody (pab2188 (IgG₁ variant) or pab2187 (IgG₁ variant)) non-reactivecell population was subjected to a final, single-cell or bulk sortingstep. After several days of cell expansion, single-cell or bulk sortedcells were again tested for binding to the polyclonal anti-TIM-3antibody and non-binding to the monoclonal antibody pab2188 (IgG₁variant) or pab2187 (IgG₁ variant) using flow cytometry.

To connect phenotype with genotype, NGS sequencing was performed on bulksorted cells expressing human TIM-3 mutants. Sequence analysis showedthat the cells that were reactive to the polyclonal anti-TIM-3 antibodybut not the monoclonal anti-TIM-3 antibody pab2188 (IgG₁ variant) orpab2187 (IgG₁ variant) expressed a human TIM-3 mutant in which position40 was mutated from a Phe to an Ala, numbered according to SEQ ID NO:79.

6.3.2 Epitope Mapping of Anti-TIM-3 Antibodies Using Hydrogen-DeuteriumExchange (HDX) Mass Spectrometry

In a first study, the interaction of pab2188 (IgG₁ variant) with humanTIM-3 was studied using hydrogen-deuterium exchange (HDX) massspectrometry.

For deglycosylation treatment, 250 μg of recombinant human TIM-3/Fcchimera (R&D Systems, Cat #2365-TM) was incubated with 4 μl of PNGase Fat 37° C. for 3 hours. The human TIM-3/Fc chimera comprises the aminoacid sequence of SEQ ID NO: 102 fused to human IgG₁.

For pepsin digestion, 6.9 μg of native or deglycosylated human TIM-3/Fcchimera in 115 μl control buffer (50 mM phosphate, 100 mM sodiumchloride, pH 7.4) was denatured by adding 115 μl of 4 M guanidinehydrochloride, 0.85 M TCEP buffer (final pH 2.5), and incubating themixture for 3 minutes at 10° C. Then, the mixture was subjected toon-column pepsin digestion using an in-house packed pepsin column andthe resultant peptides were analyzed using a UPLC-MS system comprised ofa Waters Acquity UPLC coupled to a Q EXACTIVE™ hybridquadrupole-ORBITRAP™ mass spectrometer (Thermo Scientific). The peptideswere separated on a 50 mm×1 mm C8 column with a 20.5-minute gradientfrom 2-32% solvent B (0.1% formic acid in acetonitrile). Peptideidentification was conducted through searching MS/MS data against thehuman TIM-3 sequence with Mascot software. The mass tolerance for theprecursor and product ions was 20 ppm and 0.05 Da, respectively.

10 μl native or deglycosylated human TIM-3/Fc chimera (6.9 μg), 10 μlnative human TIM-3/Fc chimera and antibody mixture (6.9 μg: 12.9 μg), or10 μl deglycosylated human TIM-3/Fc chimera and antibody mixture (6.9μg: 12.9 μg) was incubated with 105 μl deuterium oxide labeling buffer(50 mM phosphate, 100 mM sodium chloride, pD 7.4) for 0 second, 60seconds, 300 seconds, 1800 seconds, 7200 seconds, 14400 seconds, and28800 seconds. Deuterium exchange was conducted either at 10° C. fornative human TIM-3/Fc chimera and its complex with antibody or at 4° C.for deglycosylated human TIM-3/Fc chimera and its complex with antibody.Deuterium exchange was quenched by adding 115 μl of 4 M guanidinehydrochloride, 0.85 M TCEP buffer (final pH 2.5). Subsequently, thequenched sample was subjected to on-column pepsin digestion and LC-MSanalysis as described above. The mass spectra were recorded in MS onlymode. For calculation of deuterium incorporation, the mass spectra for agiven peptide were combined across the extracted ion chromatogram peaksand the weighted average m/z was calculated. Mass increase from the massof the native peptide (0 minute) to the weighted averaged masscorresponds to the level of deuterium incorporation.

The sequence coverage achieved for native and deglycosylated human TIM-3was 71.6% and 98.4%, respectively. While most human TIM-3 peptidesdisplayed identical or similar deuterium levels with and without theanti-human TIM-3 antibody, several peptide segments were found to havesignificantly decreased deuterium incorporation upon antibody binding.Both native and deglycosylated human TIM-3 showed significant reductionin deuterium uptake upon binding to anti-human TIM-3 antibody pab2188(IgG₁ variant) at a region consisting of the amino acid sequence of SEQID NO: 94 (VCWGKGACPVFECGNVVL) and a region consisting of the amino acidsequence of SEQ ID NO: 95 (RIQIPGIMND). The strongest decrease indeuterium uptake was observed at a region consisting of the amino acidsequence of SEQ ID NO: 93 (PVFECGN).

Next, the interaction of AM-2 (IgG₁ N297A) with human TIM-3 was studiedin a HDX mass spectrometry study similar to the one described above.Briefly, deglycosylated human TIM-3/Fc chimera was incubated indeuterium oxide either alone or in complex with the anti-human TIM-3antibody AM-2 (IgG₁ N297A). The deuterium exchange was carried at 10° C.for 0 second, 60 seconds, 300 seconds, 1800 seconds, 7200 seconds, and14400 seconds. The exchange reaction was quenched by low pH and thequenched samples were subjected to on-column pepsin/protease XIII orprotease XVIII digestion and LC-MS analysis as described above. Raw MSdata were processed using HDX WorkBench, software for the analysis ofHID exchange MS data (J. Am. Soc. Mass Spectrom. 2012, 23 (9),1512-1521, herein incorporated by reference in its entirety). Thedeuterium levels were calculated using the average mass differencebetween the deuterated peptide and its native form (to).

A hundred percent sequence coverage was achieved for deglycosylatedhuman TIM-3. The anti-TIM-3 antibody AM-2 (IgG₁ N297A) showed a similarbinding pattern as the one exhibited by pab2188 (IgG₁ variant). Tworegions, one consisting of the amino acid sequence of SEQ ID NO: 94(VCWGKGACPVFECGNVVL) and the other consisting of the amino acid sequenceof SEQ ID NO: 96 (RIQIPGIMNDEKFNLKL), experienced strong deuteriumprotection when deglycosylated human TIM-3 was bound to the anti-TIM-3antibody AM-2 (IgG₁ N297A). The strongest decrease was observed at aregion consisting of the amino acid sequence of SEQ ID NO: 93 (PVFECGN).

6.3.3 Epitope Mapping of Anti-TIM-3 Antibody Using Pepscan Analysis

The binding of anti-TIM-3 antibody pab2188 (IgG₁ variant) was measuredagainst synthetic TIM-3-related peptide fragments prepared as achip-bound peptide array. Analysis was performed by Pepscan Presto BV,Lelystad, the Netherlands. Briefly, to reconstruct epitopes of humanTIM-3, a library of peptides was synthesized. An amino functionalizedpolypropylene support was obtained by grafting with a proprietaryhydrophilic polymer formulation, followed by reaction witht-butyloxycarbonyl-hexamethylenediamine (BocHMDA) usingdicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) andsubsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA).Standard Fmoc-peptide synthesis was used to synthesize peptides on theamino-functionalized solid support by custom modified JANUS® liquidhandling stations (Perkin Elmer). Synthesis of structural mimics wasconducted using Pepscan's proprietary Chemically Linked Peptides onScaffolds (CLIPS) technology. CLIPS technology permits structuringpeptides into single loops, double loops, triple loops, sheet-likefolds, helix-like folds and combinations thereof. The binding ofantibody to each of the synthesized peptides was tested in aPEPSCAN-based ELISA. The peptide arrays were incubated with primaryantibody solution overnight at 4° C. After washing, the peptide arrayswere incubated with a goat anti-human HRP conjugate (Southern Biotech,Cat #2010-05) for one hour at 25° C. After washing, the peroxidasesubstrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2μl/ml of 3% H₂O₂ were added. After one hour, the color development wasmeasured and quantified with a charge coupled device (CCD)—camera and animage processing system.

The Pepscan study showed that the anti-TIM-3 antibody pab2188 (IgG₁variant) recognized stretches of human TIM-3 including a regionconsisting of the amino acid sequence of SEQ ID NO: 99 (GKGACPVFE) and aregion consisting of the amino acid sequence of SEQ ID NO: 100(DFTAAFPR).

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entiretiesand for all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

Other embodiments are within the following claims.

1-72. (canceled)
 73. An isolated antibody that specifically binds tohuman TIM-3, wherein the antibody comprises a VH comprising the CDRH1,CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence ofSEQ ID NO: 55, and a VL comprising the CDRL1, CDRL2, and CDRL3 aminoacid sequences of the VL amino acid sequence of SEQ ID NO:
 56. 74. Theisolated antibody of claim 73, wherein: (a) CDRH1 comprises the aminoacid sequence of X₁X₂X₃X₄X₅S (SEQ ID NO: 48), wherein X₁ is R, S, A, G,K, M, or T, X₂ is Q, S, A, G, R, or T, X₃ is N, Y, G, or Q, X₄ is A orQ, and X₅ is W, M, A, S, or T; (b) CDRH2 comprises the amino acidsequence of SEQ ID NO: 2; (c) CDRH3 comprises the amino acid sequence ofSEQ ID NO: 3; (d) CDRL1 comprises the amino acid sequence ofX₁ASQSVX₂SSYLA (SEQ ID NO: 52), wherein X₁ is R or G, and X₂ is absentor S; (e) CDRL2 comprises the amino acid sequence of X₁ASX₂RAT (SEQ IDNO: 53), wherein X₁ is D or G, and X₂ is N, S, or T; and (f) CDRL3comprises the amino acid sequence of QQYGSSPX₁T (SEQ ID NO: 54), whereinX₁ is L or I.
 75. The isolated antibody of claim 73, wherein: (a) CDRH1comprises the amino acid sequence of X₁X₂NAWS (SEQ ID NO: 49), whereinX₁ is R or A; and X₂ is Q or R; (b) CDRH1 comprises the amino acidsequence of X₁X₂GQX₃S (SEQ ID NO: 50), wherein X₁ is K, M, or G; X₂ is Aor S; and X₃ is S or T; or (c) CDRH1 comprises the amino acid sequenceof X₁X₂QQAS (SEQ ID NO: 51), wherein X₁ is S, R, T, or G; and X₂ is A,S, T, or G.
 76. The isolated antibody of claim 73, wherein: (a) CDRH1comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1 and 4-12; (b) CDRL1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13-16; (c) CDRL2comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 17-21; and/or (d) CDRL3 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22 and
 23. 77. Theisolated antibody of claim 73, wherein the antibody comprises a VHcomprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of a VHamino acid sequence selected from the group consisting of SEQ ID NOs:24-35, and/or a VL comprising the CDRL1, CDRL2, and CDRL3 amino acidsequences of a VL amino acid sequence selected from the group consistingof SEQ ID NOs: 36-47.
 78. The isolated antibody of claim 73, wherein theantibody comprises a heavy chain constant region selected from the groupconsisting of human IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂.
 79. Theisolated antibody of claim 78, wherein the heavy chain constant regionis: (a) a variant of a wild type human IgG heavy chain constant region,wherein the variant human IgG heavy chain constant region binds to ahuman Fc gamma receptor with lower affinity than the wild type human IgGheavy chain constant region binds to the human Fc gamma receptor,optionally wherein the human Fc gamma receptor is selected from thegroup consisting of FcγRI, FcγRII, and FcγRIII; (b) IgG₁ comprising anN297A mutation, numbered according to the EU numbering system; (c) IgG₁comprising an N297Q mutation, numbered according to the EU numberingsystem; (d) non-fucosylated IgG₁; or (e) IgG₄ comprising an S228Pmutation.
 80. The isolated antibody of claim 78, wherein the heavy chainconstant region comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 70-75.
 81. The isolated antibody of claim 73,wherein the antibody comprises a light chain constant region selectedfrom the group consisting of human kappa light chain and human lambdalight chain.
 82. The isolated antibody of claim 81, wherein the lightchain constant region comprises the amino acid sequence of SEQ ID NO: 76or
 77. 83. The isolated antibody of claim 73, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 55,and/or a VL comprising the amino acid sequence of SEQ ID NO:
 56. 84. Theisolated antibody of claim 73, wherein the antibody comprises a VHcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 24-35, and/or a VL comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 36-47.
 85. An isolatedantibody that specifically binds to human TIM-3, wherein the antibodycomprises a heavy chain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 57-68, and/or a light chaincomprising the amino acid sequence of SEQ ID NO:
 69. 86. A polypeptidecomprising a VH comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 24-35.
 87. The polypeptide of claim 86,wherein the polypeptide is an antibody heavy chain.
 88. The polypeptideof claim 87, wherein the amino acid sequence of the polypeptide consistsof an amino acid sequence selected from the group consisting of SEQ IDNOs: 57-68.
 89. A polypeptide comprising a VL comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 36-47. 90.The polypeptide of claim 89, wherein the polypeptide is an antibodylight chain.
 91. The polypeptide of claim 90, wherein the amino acidsequence of the polypeptide consists of the amino acid sequence of SEQID NO:
 69. 92. A pharmaceutical composition comprising the antibody ofclaim 73 and a pharmaceutically acceptable carrier or excipient.